Recombinase-based methods for producing expression vectors and compositions for use in practicing the same

ABSTRACT

Methods are provided for producing an expression vector. In the subject methods, donor and acceptor vectors are combined in the presence of a recombinase to produce an expression vector that includes a first and second recombinase recognition site oriented in the same direction, wherein the first and second recombination sites are able to recombine with each other. In the subject methods, one of the donor and acceptor vectors includes a single recombinase recognition site while the other includes two recombinase recognition sites. Also provided are compositions for use in practicing the subject methods, including the donor and acceptor vectors themselves, as well as systems and kits that include the same. The subject invention finds use in a variety of different applications, including the transfer or cloning of a nucleic acid of interest from a first vector into one or more expression vectors, etc.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.09/356,001 filed on Jul. 14, 1999, now abandoned; the disclosure ofwhich is herein incorporated by reference.

INTRODUCTION

1. Field of the Invention

The field of this invention is molecular biology, particularlyrecombinant DNA engineering.

2. Background of the Invention

The processes of isolating, cloning and expressing genes are central tothe field of molecular biology and play prominent roles in research andindustry in biotechnology and related fields. Until recently, theisolation and cloning of genes has been achieved in vitro usingrestriction endonucleases and DNA ligases. Restriction endonucleases areenzymes which recognize and cleave double-stranded DNA at a specificnucleotide sequence, and DNA ligases are enzymes which join fragments ofDNA together via the phosphodiester bond. A DNA sequence of interest canbe “cut” or digested into manageable pieces using a restrictionendonuclease and then inserted into an appropriate vector for cloningusing DNA ligase. However, in order to transfer the DNA of interest intoa different vector—most often a specialized expressionvector—restriction enzymes must be used again to excise the DNA ofinterest from the cloning vector, and then DNA ligase is used again toligate the DNA of interest into the chosen expression vector.

The ability to transfer a DNA of interest to an appropriate expressionvector is often limited by the availability or suitability ofrestriction enzyme recognition sites. Often multiple restriction enzymesmust be employed to remove the desired coding region. Further, thereaction conditions used for each enzyme may differ such that it isnecessary to perform the excision reaction in separate steps, or it maybe necessary to remove a particular enzyme used in an initialrestriction enzyme reaction prior to completing subsequent restrictionenzyme digestions due to buffer and/or cofactor incompatibility. Many ofthese extra steps require time-consuming purification of the subcloningintermediate.

There is, therefore, a need to develop protocols and compositions forthe rapid transfer of a DNA molecule of interest from one vector toanother in vitro or in vivo without the need to rely upon restrictionenzyme digestions.

Relevant Literature

U.S. Patents of interest include: U.S. Pat. Nos. 5,527,695; 5,744,336;5,851,808; 5,888,732; and 5,962,255. Also of interest is Liu et al.,Current Biology (1998) 8:1300-1309.

SUMMARY OF THE INVENTION

Methods are provided for producing an expression vector. In the subjectmethods, donor and acceptor vectors are combined in the presence of arecombinase to produce an expression vector that includes a first andsecond recombinase recognition site oriented in the same direction,wherein said first and said second recombinase recognition sites arecapable of recombining with each other. In the subject methods, one ofthe donor and acceptor vectors includes a single recombinase recognitionsite while the other includes two recombinase recognition sites. Alsoprovided are compositions for use in practicing the subject methods,including the donor and acceptor vectors themselves, as well as systemsand kits that include the same. The subject invention finds use in avariety of different applications.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B. FIG. 1A provides a schematic representation of a preferredembodiment of the subject methods. FIG. 1B provides the reading framespDNR-1 to pDNR-3 vectors depicted in FIGS. 2A to 2C, respectively.

FIGS. 2A to 2D provide schematic representations of four different donorplasmid vectors, i.e., pDNR-1; pDNR-2; and pDNR-3; pDNR-Lib according toa preferred embodiment of the subject invention.

FIGS. 3A to 3J provide schematic representations of 15 differentacceptor plasmids, i.e., pLP-GADT7; pLP-GBKT7; pLP-EGFP-C1; pLP-ECFP-C1;pLP-EYFP-C1; pLP-IRESneo; pLP-IRES2-EGFP; pLP-TRE2; pLP-RevTRE; andpLP-LNCX suitable for use with the donor plasmids pDNR-1; pDNR-2 andpDNR-3. Other specific acceptor vectors of interest are pLP-ProTet;pLP-CMV-Myc; pLP-CMV-HA; pLP-Shuttle; pLP-AdenoX, as described morefully in the specification.

DEFINITIONS

As used herein, the term “donor construct” refers to a donor vector,i.e., a donor nucleic acid construct comprising two donorsequence-specific recombinase target sites each having a defined 5′ to3′ orientation and placed in the donor construct such that they have thesame 5′ to 3′ orientation, and a unique restriction enzyme site orpolylinker, wherein the restriction enzyme site or polylinker is located3′ of the first-donor sequence-specific recombinase target site and 5′of the second-donor sequence-specific recombinase target site, andwherein the recombinase recognition sites are capable of recombiningwith each other.

As used herein, the term “first donor fragment” or “desired donorfragment” refers to the fragment produced when the donor construct isresolved, comprising a single sequence-specific recombinase target sitehaving a 5′ to 3′ orientation wherein the 5′ half of the singlesequence-specific recombinase target site is derived from the 5′ half ofthe second-donor sequence-specific recombinase target site in the donorconstruct and the 3′ half of the single sequence-specific recombinasetarget site is derived from the 3′ half of the first-donorsequence-specific recombinase target site of the donor construct, apolylinker or unique restriction site 3′ to said sequence-specificrecombinase target site, and the donor-partial selectable marker, or incertain embodiments, a donor-functional selectable marker. It is thefirst donor fragment that will combine with the acceptor construct toproduce the final desired recombination product.

As used herein the term “second donor fragment” or “non-desired donorfragment” refers to the fragment produced when the donor construct isresolved, comprising a single sequence-specific recombination targetsite in which the 5′ half is derived from the 5′ half of the first-donorsequence-specific recombinase target site from the donor construct andthe 3′ half is derived from the 3′ half of the second-donorsequence-specific recombinase target site from the donor construct.

As used herein, the term “acceptor construct” refers to an acceptornucleic acid construct comprising at least one origin of replication, anacceptor sequence-specific recombinase target site having a defined 5′to 3′ orientation, a first promoter located at the 5′ end of theacceptor sequence-specific recombinase target site, and in certainembodiments, an acceptor-partial selectable marker.

As used herein, “final recombination constructs” refers to therecombination products produced when either the first donor fragment orthe second donor fragment recombines with an acceptor construct, i.e.,to generate expression vectors produced by the subject methods.

As used herein, “final desired recombination construct” refers to arecombination product produced when the first, or desired, donorfragment recombines with an acceptor construct, i.e., an expressionconstruct.

The terms “sequence-specific recombinase” and “site-specificrecombinase” refer to enzymes or recombinases that recognize and bind toa short nucleic acid site or “sequence-specific recombinase targetsite”, i.e., a recombinase recognition site, and catalyze therecombination of nucleic acid in relation to these sites. These enzymesinclude recombinases, transposases and integrases.

The terms “sequence-specific recombinase target site”, “site-specificrecombinase target site”, “sequence-specific target site” and“site-specific target site” refer to short nucleic acid sites orsequences, i.e., recombinase recognition sites, which are recognized bya sequence- or site-specific recombinase and which become the crossoverregions during a site-specific recombination event. Examples ofsequence-specific recombinase target sites include, but are not limitedto, lox sites, att sites, dif sites and frt sites.

The term “lox site” as used herein refers to a nucleotide sequence atwhich the product of the cre gene of bacteriophage P1, the Crerecombinase, can catalyze a site-specific recombination event. A varietyof lox sites are known in the art, including the naturally occurringloxP, loxB, loxL and loxR, as well as a number of mutant, or variant,lox sites, such as loxP511, loxP514, loxΔ86, loxΔ117, loxC2, loxP2,loxP3 and lox P23.

The term “frt site” as used herein refers to a nucleotide sequence atwhich the product of the FLP gene of the yeast 2 micron plasmid, FLPrecombinase, can catalyze site-specific recombination.

The term “unique restriction enzyme site” indicates that the recognitionsequence of a given restriction enzyme appears once within a nucleicacid molecule.

A restriction enzyme site or restriction site is said to be located“adjacent to the 3′ end of a sequence-specific recombinase target site”if the restriction enzyme recognition site is located downstream of the3′ end of the sequence-specific recombinase target site. The adjacentrestriction enzyme site may, but need not, be contiguous with the lastor 3′ most nucleotide comprising the sequence-specific recombinasetarget site.

The terms “polylinker” or “multiple cloning site” refer to a cluster ofrestriction enzyme sites, typically unique sites, on a nucleic acidconstruct that can be utilized for the insertion and/or excision ofnucleic acid sequences, such as the coding region of a gene, loxP sites,etc.

The term “termination sequence” refers to a nucleic acid sequence whichis recognized by the polymerase of a host cell and results in thetermination of transcription. Prokaryotic termination sequences commonlycomprise a GC-rich region that has a two-fold symmetry followed by anAT-rich sequence. A commonly used termination sequence is the T7termination sequence. A variety of termination sequences are known inthe art and may be employed in the nucleic acid constructs of thepresent invention, including the TINT3, TL13, TL2, TR1, TR2, and T6Stermination signals derived from the bacteriophage lambda, andtermination signals derived from bacterial genes, such as the tip geneof E. coli.

The terms “polyadenylation sequence” (also referred to as a “poly A⁺site” or “poly A⁺ sequence”) as used herein denotes a DNA sequence whichdirects both the termination and polyadenylation of the nascent RNAtranscript. Efficient polyadenylation of the recombinant transcript isdesirable, as transcripts lacking a poly A⁺ tail are typically unstableand rapidly degraded. The poly A⁺ signal utilized in an expressionvector may be “heterologous” or “endogenous”. An endogenous poly A⁺signal is one that is found naturally at the 3′ end of the coding regionof a given gene in the genome. A heterologous poly A⁺ signal is onewhich is isolated from one gene and placed 3′ of another gene, e.g.,coding sequence for a protein. A commonly used heterologous poly A⁺signal is the SV40 poly A⁺ signal. The SV40 poly A⁺ signal is containedon a 237 bp BamHI/BclI restriction fragment and directs both terminationand polyadenylation; numerous vectors contain the SV40 poly A⁺ signal.Another commonly used heterologous poly A⁺ signal is derived from thebovine growth hormone (BGH) gene; the BGH poly A⁺ signal is alsoavailable on a number of commercially available vectors. The poly A⁺signal from the Herpes simplex virus thymidine kinase (HSV tk) gene isalso used as a poly A⁺ signal on a number of commercial expressionvectors.

As used herein, the terms “selectable marker” or “selectable markergene” refer to a gene which encodes an enzymatic activity and confersthe ability to grow in medium lacking what would otherwise be anessential nutrient; in addition, a selectable marker may confer upon thecell in which the selectable marker is expressed, resistance to anantibiotic or drug. A selectable marker may be used to confer aparticular phenotype upon a host cell. When a host cell must express aselectable marker to grow in selective medium, the marker is said to bea positive selectable marker (e.g., antibiotic resistance genes whichconfer the ability to grow in the presence of the appropriateantibiotic). Selectable markers can also be used to select against hostcells containing a particular gene; selectable markers used in thismanner are referred to as negative selectable markers.

As used herein, the term “donor-partial selectable marker” found incertain embodiments of the subject invention refers to portion of aselectable marker gene encoded by the donor construct which isnon-functional by itself, by which is meant that it must be positionedon a vector in operable relation with another element in order to beexpressed. Examples of donor-partial selectable markers are codingsequences and promoter regions of complete selectable markers offunctioning expression modules or cassettes.

As used herein, the term “donor-functional selectable marker” found incertain embodiments of the subject invention refers to a functionalselectable marker gene encoded by the donor construct.

As used herein, the term “acceptor-partial selectable marker” found incertain embodiments of the subject invention refers to a portion of aselectable marker gene encoded by the acceptor construct which isnon-functional by itself, as described above, e.g., a coding sequence orpromoter by itself

As used herein, the term “acceptor-functional selectable marker” foundin certain embodiments of the subject invention refers to a functionalselectable marker gene encoded by the acceptor construct.

As used herein, the term “recombinant-functional selectable marker”refers to the functional selectable marker gene created uponrecombination between the donor construct and the acceptor constructwhich results in the adjacent placement of the donor-partial selectablemarker and the acceptor-partial selectable marker, i.e., flanking eitherside of a recombinase site.

As used herein, the term “construct” is used in reference to nucleicacid molecules that transfer DNA segment(s) from one cell to another.The term “vector” is sometimes used interchangeably with “construct”.The term “construct” includes circular nucleic acid constructs such asplasmid constructs, phagemid constructs, cosmid vectors, etc., as wellas linear nucleic acid constructs including, but not limited to, PCRproducts. The nucleic acid construct may comprise expression signalssuch as a promoter and/or an enhancer in operable linkage, and then isgenerally referred to as an “expression vector” or “expressionconstruct”.

The term “expression construct” as used herein refers to an expressionmodule or expression cassette made up of a recombinant DNA moleculecontaining a desired coding sequence and appropriate nucleic acidsequences necessary for the expression of the operably linked codingsequence in a particular host organism. Nucleic acid sequences necessaryfor expression in prokaryotes usually include a promoter and a ribosomebinding site, often along with other sequences. Eukaryotic cells areknown to utilize promoters, enhancers, and termination andpolyadenylation signals.

The terms “in operable combination”, “in operable order” and “operablylinked” as used herein refer to the linkage of nucleic acid sequences insuch a manner that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced. The terms also refer to the linkage of amino acidsequences in such a manner so that the reading frame is maintained and afunctional protein is produced.

A cell has been “transformed” or “transfected” with exogenous orheterologous DNA when such DNA has been introduced inside the cell. Thetransforming DNA may or may not be integrated (covalently linked) intothe genome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a vector or plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA is inherited bydaughter cells through chromosome replication. This stability isdemonstrated by the ability of the eukaryotic cell to establish celllines or clones comprised of a population of daughter cells containingthe transforming DNA. A “clone” is a population of cells derived from asingle cell or ancestor by mitosis. A “cell line” is a clone of aprimary cell that is capable of stable growth in vitro for manygenerations. An organism, such as a plant or animal, that has beentransformed with exogenous DNA is termed “transgenic”.

Transformation of prokaryotic cells may be accomplished by a variety ofmeans known in the art, including the treatment of host cells with CaCl₂to make competent cells, electroporation, etc. Transfection ofeukaryotic cells may be accomplished by a variety of means known in theart, including calcium phosphate-DNA co-precipitation,DEAE-dextran-mediated transfection, polybrene-mediated transfection,electroporation, microinjection, liposome fusion, lipofection,protoplast fusion, retroviral infection, and biolistics.

As used herein, the term “host” is meant to include not onlyprokaryotes, but also eukaryotes, such as yeast, plant and animal cells.A recombinant DNA molecule or gene can be used to transform a host usingany of the techniques commonly known to those of ordinary skill in theart. Prokaryotic hosts may include E. coli, S. tymphimurium, Serratiamarcescens and Bacillus subtilis. Eukaryotic hosts include yeasts suchas Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris,mammalian cells and insect cells, and, plant cells, such as Arabidopsisthaliana and Tobaccum nicotiana.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

“Recombinant DNA technology” refers to techniques for uniting twoheterologous DNA molecules, usually as a result of in vitro ligation ofDNAs from different organisms. Recombinant DNA molecules are commonlyproduced by experiments in genetic engineering. Synonymous terms include“gene splicing”, “molecular cloning” and “genetic engineering”. Theproduct of these manipulations results in a “recombinant” or“recombinant molecule”. The term “recombinant protein” or “recombinantpolypeptide” as used herein refers to a protein molecule which isexpressed from a recombinant DNA molecule.

The ribose sugar is a polar molecule, and therefore, DNA is referred toas having a 5′ to 3′, or 5′ to 3′, directionality. DNA is said to have“5′ ends” and “3′ ends” because mononucleotides are reacted to makeoligonucleotides in a manner such that the 5′ phosphate of onemononucleotide pentose ring is attached to the 3′ oxygen of its neighborvia a phosphodiester linkage. Therefore, an end of an oligonucleotide isreferred to as the “5′ end” if its 5′ phosphate is not linked to the 3′oxygen of a mononucleotide pentose ring and as the “3′ end” if its 3′oxygen is not linked to a 5′ phosphate of a subsequent mononucleotidepentose ring. As used herein, a nucleic acid sequence, even if internalto a larger oligonucleotide, also has a 5′ to 3′ orientation. In eithera linear or circular DNA molecule, discrete elements are referred to asbeing “upstream” or “5′” of the “downstream” or “3′” elements. Thisterminology reflects the fact that DNA has an inherent 5′ to 3′polarity, and transcription typically proceeds in a 5′ to 3′ fashionalong the DNA strand. The promoter and enhancer elements which directtranscription of an operably linked coding region, or open readingframe, are generally located 5′, or upstream, of the coding region.However, enhancer elements can exert their effect even when located 3′of the promoter and coding region. Transcription termination andpolyadenylation signals are typically located 3′ or downstream of thecoding region.

The 3′ end of a promoter is said to be located upstream of the 5′ end ofa sequence-specific recombinase target site when, moving in a 5′ to 3′direction along the nucleic acid molecule, the 3′ terminus of a promoterprecedes the 5′ end of the sequence-specific recombinase target site.When the acceptor construct is intended to permit the expression of atranslation fusion, the 3′ end of the promoter is located upstream ofboth the sequences encoding the amino-terminus of a fusion protein andthe 5′ end of the sequence-specific recombinase target site. Thus, thesequence-specific recombinase target site is located within the codingregion of the fusion protein (i.e., located downstream of both thepromoter and the sequences encoding the affinity domain, such as Gst).

As used herein, the term “adjacent”, in the context of positioning ofgenetic elements in the constructs, shall mean within about 0 to 2500,sometimes 0 to 1000 bp and sometimes within about 0 to 500, 0 to 400, 0to 300 or 0 to 200 bp.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence. A “cDNA” is defined ascopy-DNA or complementary-DNA, and is a product of a reversetranscription reaction from an mRNA transcript. An “exon” is anexpressed sequence transcribed from the gene locus, whereas an “intron”is a non-expressed sequence that is from the gene locus.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell. A “cis-element” is a nucleotide sequence, alsotermed a “consensus sequence” or “motif”, that interacts with proteinsthat can upregulate or downregulate expression of a specific gene locus.A “signal sequence” can also be included with the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell and directs the polypeptide to theappropriate cellular location. Signal sequences can be found associatedwith a variety of proteins native to prokaryotes and eukaryotes.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence includes, at its 3′ terminus, thetranscription initiation site and extends upstream (in the 5′ direction)to include the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site, as well asprotein binding domains (consensus sequences) responsible for thebinding of RNA polymerase. Eukaryotic promoters often, but not always,contain “TATA” boxes and “CAT” boxes.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires expression of signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length.

As used herein, “an origin of replication” or “origin” refers to anysequence capable of directing replication of a DNA construct in asuitable prokaryotic or eukaryotic host (e.g., the ColE1 origin and itsderivatives; the yeast 2μ origin). Eukaryotic expression vectors mayalso contain “viral replicons” or “origins of replication”. Viralreplicons are viral DNA sequences which allow for the extrachromosomalreplication of a vector in a host cell expressing the appropriatereplication factors. Vectors which contain either the SV40 or polyomavirus origin of replication replicate to high copy number (up to 10⁴copies/cell) in cells that express the appropriate viral T antigen.Vectors which contain the replicons from bovine papillomavirus orEpstein-Barr virus replicate extrachromosomally at low copy number (˜100copies/cell).

As used herein, the terms “nucleic acid molecule encoding”, “DNAsequence encoding”, and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

As used herein, the term “gene” means the deoxyribonucleotide sequencescomprising the coding region of a structural gene, i.e., the codingsequence for a protein or polypeptide of interest, including sequenceslocated adjacent to the coding region on both the 5′ and 3′ ends for adistance of about 1 kb on either end, such that the gene corresponds tothe length of the full-length mRNA. The sequences which are located 5′of the coding region and which are present on the mRNA are referred toas 5′ non-translated sequences. The sequences which are located 3′ ordownstream of the coding region and which are present on the mRNA arereferred to as 3′ non-translated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene. A genomic form or clone of a genecontains the coding region interrupted with non-coding sequences termed“introns” or “intervening regions” or “intervening sequences”. Intronsare segments of a gene which are transcribed into heteronuclear RNA(hnRNA); introns may contain regulatory elements such as enhancers.Introns are removed or “spliced out” from the nuclear or primarytranscript; introns therefore are absent in the mature messenger RNA(mRNA) transcript. The mRNA functions during translation to specify thesequence or order of amino acids in a nascent polypeptide.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequenceswhich are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers which control or influence thetranscription of the gene. The 3′ flanking region may contain sequenceswhich direct the termination of transcription, post-transcriptionalcleavage and polyadenylation.

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample. For example, recombinant Cre polypeptidesare expressed in bacterial host cells (e.g., as a GST-Cre or (HN)₆-Crefusion protein) and the Cre polypeptides are purified by the removal ofhost cell proteins; the percent of recombinant Cre polypeptides isthereby enriched or increased in the sample.

As used herein the term “portion” refers to a fraction of a sequence,gene or protein. “Portion” may comprise a fraction greater than half ofthe sequence, gene or protein, equal to half of the sequence, gene orprotein or less than half of the sequence, gene or protein. Typically asused herein, two or more “portions” combine to comprise a wholesequence, gene or protein.

As used herein, the term “fusion protein” refers to a chimeric proteincontaining a protein of interest joined to an exogenous proteinfragment. The fusion partner may enhance solubility of the protein ofinterest as expressed in a host cell, may provide an affinity tag toallow purification of the recombinant fusion protein from the host cellor culture supernatant, or both. If desired, the fusion protein may beremoved from the protein of interest by a variety of enzymatic orchemical means known to the art.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods are provided for producing an expression vector. In the subjectmethods, donor and acceptor vectors are combined in the presence of arecombinase to produce an expression vector that includes a first andsecond recombinase recognition site oriented in the same direction. . Inthe subject methods, one of the donor and acceptor vectors includes asingle recombinase recognition site while the other includes tworecombinase recognition sites. Also provided are compositions for use inpracticing the subject methods, including the donor and acceptor vectorsthemselves, as well as systems and kits that include the same. Thesubject invention finds use in a variety of different applications.

Before the subject invention is further described, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Methods

As summarized above, the subject invention provides recombinase-basedmethods for producing expression vectors. More specifically, the subjectinvention provides methods for producing expression vectors by combininga donor and acceptor vector that each include one or more recombinaserecognition sites with a recombinase under conditions sufficient forrecombinase mediated site specific recombination to occur, where suchrecombination results in the production of an expression vector thatlacks at least a portion of the donor or acceptor vector from which itis produced, i.e., to produce a non-fusion expression vector.

A feature of the subject invention is that the donor and acceptorvectors must be able to recombine in the presence of a suitablerecombinase to produce an expression vector as described above, wherethe expression vector lacks at least a portion of the initial donor oracceptor vector, i.e., it is a non-fusion expression vector. As such,the donor and acceptor vectors must be able to participate in arecombination event that is other than a fusion event, where by fusionevent is meant an event in which two complete vectors are fused in theirentirety into one fused vector, e.g., where two plasmids are fusedtogether to produce one plasmid that includes all of material from theinitial two plasmids, i.e., a fusion plasmid. As such, the subjectmethods are not fusion methods, where such methods are defined as thosemethods in which a single vector is produced from two or more initialvectors in their entirety, such that all of the initial vector materialof each parent vector, e.g., plasmid, is present in its entirety in theresultant fusion vector.

The donor and acceptor vectors are further characterized in that one ofthe donor and acceptor vectors includes only one recombinase recognitionsite, while the other of the donor and acceptor vectors includes tworecombinase recognition sites. In a first preferred embodiment, thedonor vector includes two recombinase recognition sites while theacceptor vector includes a single recombinase recognition site. In analternative embodiment, the donor vector includes a single recombinaserecognition site while the acceptor vector includes two recombinaserecognition sites. The donor and acceptor vectors of this first,preferred embodiment and this second, alternative embodiment, aredescribed in greater detail below.

The donor and acceptor vectors described generally above may be linearor circular, e.g., plasmids, and in many embodiments of the subjectinvention are plasmids. Where the donor and acceptor vectors areplasmids, the donor and acceptor vectors typically range in length fromabout 0.2 kb to 200 kb, usually from about 2 kb to 40 kb and moreusually from about 2 kb to 10 kb.

The donor and acceptor vectors are further characterized in that all ofthe recombinase recognition sites on the donor and acceptor vectors mustbe recognized by the same recombinase and should be able to recombinewith each other, but within this parameter they may be the same ordifferent, but in many embodiments are usually the same. Recombinaserecognition sites, i.e., sequence-specific recombinase target sites, ofinterest include: Cre recombinase activity recognized sites, e.g., loxP,loxP2, loxP51 1, loxP514, loxB, loxC2, loxL, loxR, lox≢86, loxΔ117; att,dif; frt; and the like. The particular recombinase recognition site ischosen, at least in part, based on the nature of the recombinase to beemployed in the subject methods.

The Donor Vector

As mentioned above, in a preferred embodiment of the subject methods,the donor vector includes two recombinase recognition sites while theacceptor vector includes a single recombinase recognition site. In thedonor vector of these embodiments, the donor vector includes tworecombinase recognition sites, capable of recombining with each other,e.g., site 1A and site 1B, that flank or border a first or donor domain,i.e., desired donor fragment, where this domain is the portion of thevector that becomes part of the expression vector produced by thesubject methods. The length of the donor domain may vary, but in manyembodiments ranges from 1 kb to 200 kb, usually from about 1 kb to 10kb. The portion of the donor vector that is not part of this donordomain, i.e., the part that is 5′ of site 1A and 3′ of site 1B, isreferred to herein for clarity as the non-donor domain of the donorvector.

The two recombinase recognition sites of the donor vector arecharacterized in that they are oriented in the same direction and arecapable of recombining with each other. By oriented in the samedirection it is meant that they have the same head to tail orientation.Thus, the orientation of site 1A is the same as the orientation of site1B.

The donor domain flanked by the two recombinase recognition sites, i.e.,the portion of the vector 3′ of the first recombinase site 1A and 5′ ofthe second recombinase site 1B, includes at least the followingcomponents: (a) at least one restriction site and (b) at least a portionof a selectable marker, e.g. a coding sequence, a promoter, or acomplete selectable marker made up of a coding sequence and a promoter.The donor domain may include at least one restriction site or aplurality of distinct restriction sites, e.g., as found in a multiplecloning site or polylinker, where by restriction site is meant a stretchof nucleotides that has a sequence that is recognized and cleaved by arestriction endonuclease. Where a plurality of restriction sites arepresent in the donor domain, the number of distinct or differentrestriction sites typically ranges from about 2 to 5, usually from about2 to 13.

In many embodiments, there are at least two restriction sites, which mayor may not be identical depending on the particular protocol employed toproduce the donor plasmid, that flank a nucleic acid which is a codingsequence for a protein of interest, where the protein of interest may ormay not be known, e.g., it may be a known coding sequence for a knownprotein or polypeptide or a coding sequence for an as yet unidentifiedprotein or polypeptide, such as where this nucleic acid of interest is aconstituent of a library, as discussed in greater detail below. Thelength of this nucleic acid of interest nucleic acid may vary greatly,but generally ranges from about 18 bp to 20 kb, usually from about 100bp to 10 kb and more usually from about 1 kb to 3 kb. At least onerestriction site and this nucleic acid of interest nucleic acid, whenpresent, are sufficiently close to the 3′ end of the first flankingrecombinase site, i.e., recombinase recognition site 1A, such that inthe expression vector produced from the donor plasmid, expression of thecoding sequence of the nucleic acid of interest is driven by a promoterpositioned 5′ of this first recombinase site. As such, the distanceseparating this restriction site/nucleic acid of interest nucleic acidfrom the recombinase site typically ranges from about 1 bp to 150 bp,usually from about 1 bp to 50 bp.

In a first preferred embodiment, the donor domain also generallyincludes a portion of a selectable marker. By portion of a selectablemarker is meant a sub-part of a selectable marker, e.g. a codingsequence or a promoter, which can be joined with a second subpart toproduce a functioning selectable marker that confers some selectablephenotype on the host cell in which the expression vector produced bythe subject methods is to be propagated. Examples of subparts ofselectable markers are coding sequences and promoters. As such, in manyembodiments, the portion of the selectable marker present on the donordomain is a coding sequence of a marker gene or a promoter capable ofdriving expression of the coding sequence of the marker gene, where incertain preferred embodiments, the coding sequence of a marker gene isthe portion of the selectable marker present on the donor domain.Examples of coding sequences of interest include, but are not limitedto, the coding sequences from the following marker genes:_thechloramphenicol resistance gene, the ampicillin resistance gene, thetetracycline resistance gene, the kanamycin resistance gene, thestreptomycin resistance gene and the SacB gene from B. subtilis encodingsucrase and conferring sucrose sensitivity; and the like. The promoterportions or sub-parts of this selectable marker are any convenientpromoters capable of driving expression of the selectable marker in theexpression vector produced by the subject methods, see infra, and inmany embodiments are bacterial promoters, where particular promoters ofinterest include, but are not limited to: the Ampicillin resistancepromoter, the inducible lac promoter, the tet-inducible promoter frompProTet (P_(ltetO-1))-available from CLONTECH, T7, T3, and SP6promoters; and the like. The distance of this sub-part or portion of theselectable marker from the 3′ end of the second recombinase recognitionsite, i.e., site 1B, is sufficient to provide for expression of themarker to occur in the final expression vector, where the other part ofselectable marker that is required for efficient expression of theselectable marker is present on the other side, i.e., the 5′ side of theadjacent recombinase recognition site. This distance typically rangesfrom about 1 bp to 2.5 kb, usually from about 1 bp to 500 bp.

The length of the donor domain flanked by the first and secondrecombinase sites of the donor plasmid, i.e., the length of the desireddonor fragment, may vary greatly, so long as the above describedcomponents are present on the donor domain. Generally, the length is atleast about 100 bp, usually at least about 500 bp and more usually atleast about 900 bp, where the length may be as great as 100 kb orgreater, but generally does not exceed about 20 kb and usually does notexceed about 10 kb. Typically, the length of the donor domain rangesfrom about 100 bp to 100 kb, usually from about 500 bp to 20 kb and moreusually from about 900 bp to 10 kb.

In addition to the above described components, the donor vector mayinclude a number of additional elements, where desired, that are presenton the non-donor domain or non-desired donor fragment of the donorvector. For example, the non-donor domain generally includes an originof replication. This origin of replication may be any convenient originof replication or ori site, where a number of ori sites are known in theart, where particular sites of interest include, but are not limited to:ColE1 and its derivatives, pMB 1, other origins that function inprokaryotic cells, the yeast 2 micron origin and the like. Also presenton this non-donor domain of certain preferred embodiments is a selectivemarker gene that provides for negative selection of the non-donor domainunder particular conditions, e.g., negative selection conditions. Thismarker is fully functional and therefor is made up of a coding sequenceoperably linked to an appropriate promoter, i.e., is provided by afunctional expression module or cassette. Markers of interest that arecapable of providing for this negative selection include, but are notlimited to: SacB, providing sensitivity to sucrose; ccdB; and the like.

This non-donor domain of the donor vector may further include one ormore additional components or elements that impart additionalfunctionality to the donor vector. For example, the donor vector may bea vector that is specifically designed for use in conjunction with ayeast two hybrid assay protocol, e.g., such that one can determinewhether the gene of interest present in the donor domain encodes aproduct that binds to a second protein prior to transferal of the geneof interest to an expression vector. In such embodiments, the non-donordomain typically includes the following additional elements: yeastorigins of replication, e.g., the yeast 2 micron origin; yeast selectionmarkers, e.g., URA3, Leu, and trp selection markers; and peptidefragments of yeast transcription factors that are expressed astranslational fusions to the gene encoded within the donor-domain; whereyeast two hybrid systems are known to those of skill in the art anddescribed in: Fields, S. and O-K. Song. 1989. A novel genetic system todetect protein-protein interactions. Nature 340:245-246; Fields, S. andR. Sternglanz. 1994. The two-hybrid system: an assay for protein-proteininteractions. Trends Genet. 10: 286-292 and the MATCHMAKER system IIIuser manual, available from CLONTECH. In other embodiments, thenon-donor domain main contain yet other functional elements that providespecific functions to the donor. For example, Donor vectors can bedesigned that would also function as prokaryotic expression vectors thatexpress the gene of interest encoded on the donor domain in prokaryoticcells either as a native protein or fused to an affinity or epitope tag.Such vectors may include the following elements in their non-donordomain: inducible bacterial promoters, such as the lac promoter or theP_(ltetO-1) promoter; affinity or epitope tags, e.g., GST, 6x(HN),myc-tag, HA-Tag, GFP and its derivatives. Donor vectors designed tofunction as retroviral vectors would additionally include retroviralLTRs and packaging signals in the non-donor domain. Donor vectors forexpression in mammalian cells might also encode affinity or epitopetags, e.g., GST, 6x(HN), myc-tag, HA-Tag, GFP and its derivatives; andmammalian constitive or inducible promoters, e.g., the CMV promoter, thetet-inducible promoter, the TK promoter; viral promoters, e.g., T7, T3,SP6. In a preferred embodiment of this particular embodiment of thesubject invention, the donor vector is as follows. The donor-partialselectable marker comprises the open reading frame (ORF) for aselectable marker gene, and is placed between the two donorsequence-specific recombinase target sites, adjacent to the second-donorsequence-specific recombinase target site. In a more preferredembodiment of the donor construct, the open reading frame of theselectable marker is situated such that its 5′ to 3′ orientation isopposite that of the two donor sequence-specific recombinase targetsites.

In another embodiment of the donor construct, the donor construct is aclosed circle (e.g., a plasmid or cosmid) comprising, in addition to thetwo donor sequence-specific recombinase target sites, the uniquerestriction site or polylinker and the selectable marker gene openreading frame, at least one origin of replication, and at least onedonor-functional selectable marker gene. The methods of the presentinvention should not be limited by the origin of replication selected.For example, origins such as those found in the pUC series of plasmidvectors or of the pBR322 plasmid may be used, as well as others known inthe art. Those skilled in the art know that the choice of origin dependson the application for which the donor construct is intended and/or thehost strain in which the construct is to be propagated.

A variety of selectable marker genes may be utilized, either for thedonor-partial selectable marker or for the donor-functional selectablemarker, and such genes may confer either positive- ornegative-resistance phenotypes; however, the donor-partial and thedonor-functional selectable marker genes should be different from oneanother. In a preferred embodiment, the selectable markers are selectedfrom the group consisting of the chloramphenicol resistance gene, theampicillin resistance gene, the tetracycline resistance gene, thekanamycin resistance gene, the streptomycin resistance gene and the sacBgene from B. subtilis encoding sucrase and conferring sucrosesensitivity. In a more preferred embodiment, the donor-partialselectable marker is a portion of the gene (e.g., the open readingframe) for chloramphenicol resistance and the donor-functionalselectable marker gene is the gene for ampicillin resistance . Inanother preferred embodiment of the donor construct, the origin ofreplication and the donor-functional selectable marker gene lie 5′ ofthe first-donor sequence-specific recombinase target site.

In another embodiment of the present invention, there is provided adonor construct with all the above-described features, but additionallyhaving a marker gene different from either the donor-functionalselectable marker gene or the donor-partial selectable marker gene,wherein the additional marker gene is positioned 5′ of the firstsequence-specific recombinase target site such that upon combinationwith a recombinase, the additional marker gene is located on theundesired second donor fragment. This marker gene provides an additionalscreen to exclude any products that result in recombinants containingthe second donor fragment. The marker gene could be, for example, LacZ.In this case, incorrect recombinants would generate blue colonies onX-Gal plates. Alternatively, a more preferred additional marker would bethe sacB gene conferring sucrose sensitivity. In this case, anyincorrect clones would be killed when grown on sucrose containingmedium. The additional marker provides another screen, thereby enhancingthe system by further ensuring that only correct recombination productsare obtained following recombination and transformation.

In yet another embodiment of the donor construct, the donor constructfurther comprises a termination sequence placed 3′ of the restrictionsite or polylinker sequence but 5′ of the second-donor sequence-specificrecombinase target site. In a most preferred embodiment, the terminationsequence is placed 5′ of the 3′ end of the donor-partial selectablemarker (e.g. the ORF of the selectable marker gene in the preferredembodiment which is in the 5′ to 3′ orientation opposite that of bothdonor sequence specific recombinase target sites). The presentembodiment is not be limited by the termination sequence chosen. In oneembodiment, the termination sequence is the T1 termination sequence;however, a variety of termination sequences are known to the art and maybe employed in the nucleic acid constructs of the present invention,including the T6S, TINT, TL1, TL2, TR1, and TR2 termination signalsderived from the bacteriophage lambda, and termination signals derivedfrom bacterial genes such as the trp gene of E. coli.

In another preferred embodiment of the donor construct, the donorconstruct further comprises a polyadenylation sequence placed 3′ of theunique restriction site(s) or polylinker but 5′ of the second-donorsequence-specific recombinase target site. In a most preferredembodiment, the polyadenylation sequence is placed 5′ of the 3′ end ofthe open reading frame of the selectable marker gene similar to theplacement described for the termination sequence supra. The presentinvention should not be limited by the nature of the polyadenylationsequence chosen. In one embodiment, the polyadenylation sequence isselected from the group consisting of the bovine growth hormonepolyadenylation sequence, the simian virus 40 polyadenylation sequenceand the Herpes simplex virus thymidine kinase polyadenylation sequence.

Also, in a preferred embodiment, the donor construct further comprises agene or DNA sequence of interest inserted into the unique restrictionenzyme site or polylinker. The present invention should not be limitedby the size of the DNA of interest inserted into the unique restrictionsite or polylinker nor the source of DNA (e.g., genomic libraries, cDNAlibraries, etc.).

Thus, in a most preferred embodiment of the donor nucleic acidconstruct, there is provided, in 5′ to 3′ order: a) a first-donorsequence-specific recombinase target site; b) a nucleic acid or gene ofinterest; c) termination and polyadenylation sequences; d) an openreading frame for a selectable marker gene in a 5′ to 3′ orientationopposite to that of the first-donor sequence-specific recombinase targetsite; e) a second-donor sequence-specific recombinase target site in thesame 5′ to 3′ orientation as the first donor sequence-specificrecombinase target site, wherein the second-donor sequence-specificrecombinase target site is able to recombine with said first-donorsequence-specific recombinase target site; f) an origin of replication;and g) a donor-functional selectable marker gene.

As mentioned above, in an alternative embodiment of the subjectinvention, the donor vector employed in the subject methods includesonly a single recombinase recognition site, while the acceptor vector,described in greater detail below, includes two recombinase recognitionsites. In this embodiment, the donor vector includes: a) a donor partialselectable marker element; b) one sequence-specific recombinase targetsite with a defined 5′ to 3′ orientation; and c) a unique restrictionenzyme site or polylinker, said restriction enzyme site or polylinkerbeing located 3′ of the sequence-specific recombinase target site. Thedonor partial selectable marker element must be placed in said donorconstruct so that when the donor construct later recombines with theacceptor construct, a functional selectable marker is formed in theresulting final recombination product. In a preferred embodiment of thisalternative embodiment, the donor partial selectable marker elementcomprises the open reading frame (ORF) for a selectable marker geneplaced adjacent to the sequence-specific recombination site such thatits 5′ to 3′ orientation is opposite to that of the sequence-specificrecombination site. In addition, in this preferred embodiment of thealternative embodiment of the donor construct, the donor construct is aclosed circle (e.g., a plasmid or cosmid) comprising, in addition tosaid sequence-specific recombinase target site, said unique restrictionsite or polylinker and said selectable marker gene open reading frame,an origin of replication capable of replicating the final recombinationconstruct, a functional selectable marker gene driven by a promoter, aprokaryotic termination sequence placed 3′ of the restriction site orpolylinker sequence and a eukaryotic polyadenylation sequence placed 3′of the restriction site or polylinker. Also, in a preferred embodimentof the alternative embodiment, the donor construct further comprises agene or DNA sequence of interest inserted into the unique restrictionenzyme site or polylinker. The present invention should not be limitedby the size of the DNA of interest inserted into the unique restrictionsite or polylinker.

FIGS. 2A to 2C provide schematic representations of three differentrepresentative specific donor vectors, specifically donor plasmids, ofthe subject invention, i.e., pDNR1; pDNR2 and pDNR3. Each of thesespecific representative vectors includes two loxP sites oriented in thesame direction. Also present in each of these specific donor plasmids isthe chloramphenicol resistance open reading frame and a multiple cloningsite, which elements are flanked by the lox P sites and are present onthe part of the plasmid that is incorporated into the final expressionvector upon practice of the subject methods. Also present on each of thedonor plasmids are two selectable markers on the portion of the plasmidthat is not incorporated into the final expression vector, i.e., Amp^(r)and SacB. These three specific donor plasmids differ from each otherwith respect to the multiple cloning site, and specifically the openreading frame of the multiple cloning site, as shown in FIG. 1B. Yetanother specific donor vector of interest is the p-DNR-Lib vector, shownin FIG. 2D. In this vector only one selectable marker is present on theportion of the plasmid that is not incorporated into the finalexpression vector, e.g., SacB.

The Acceptor Vector

As mentioned above, in a preferred embodiment of the subject invention,the acceptor vector employed in the subject methods is a vector thatincludes a single recombinase site. In these embodiments, the singlerecombinase site is flanked on one side by a promoter and on the otherside, in certain preferred embodiments, by a portion of a selectablemarker, e.g., a promoter or a coding sequence, where in many preferredembodiments described further below, this portion or sub-part of theselectable marker is a second promoter, e.g., a bacterial promoter. Inthese embodiments, the single recombinase site is flanked by twooppositely oriented promoters, where one of promoters drives expressionof the gene of interest in the expression vector produced by the subjectmethods and the second promoter drives expression of the coding sequenceof the recombinant-functional selectable marker in the expression vectorproduced by the subject methods. In these embodiments, the firstpromoter is a promoter that is capable of driving expression of the geneof interest in the expression vector, where representative promotersinclude, but are not limited to the CMV promoter, the tet-induciblepromoter; retroviral LTR promoter/enhancer sequences, the TK promoter,bacterial promoters, e.g. the lac promoter, the P_(LtetO-1) promoter;the yeast ADH promoter and the like. The distance between the firstpromoter and the recombinase site is one that allows for expression inthe final expression vector, where the distance typically ranges fromabout 1 bp to 1000 bp, usually from about 10 bp to 500 bp. The secondpromoter is a promoter that is capable of driving expression of therecombinant-functional selectable marker, and is generally a bacterialpromoter. Bacterial promoters of interest include, but are not limitedto: the Ampicillin promoter, the lac promoter , the P_(LtetO-1) promoter, the T7 promoter and the like. The distance between the bacterialpromoter and the recombinase site is sufficient to provide forexpression of the selectable marker in the expression vector andtypically ranges from about 1 bp to 2.5 kb, usually from about 1 bp to200 bp.

As indicated above, in yet other preferred embodiments the acceptorvector lacks the portion or subpart of the selectable marker. In theseembodiments, the acceptor vector may be used with a donor vector thatincludes a complete positive selectable marker in the desired donorfragment flanked by the two recombinase sites, i.e., the donor vectorportion located between the 3′ end of the first recombinase site and the5′ end of the second recombinase site. Alternatively, the acceptorvector may be used with a donor vector that only includes a partialselectable positive marker, as described above, where the partial markeris nonetheless functional in the resultant expression vector.

The acceptor vector of the embodiments described above may include anumber of additional components or elements which are requisite ordesired depending on the nature of the expression vector to be producedfrom the acceptor vector. In many embodiments of the subject invention,the acceptor vector is an acceptor nucleic acid construct comprising: a)an origin of replication capable of replicating the final desiredrecombination construct or expression vector; b) an acceptorsequence-specific recombinase target site having a defined 5′ to 3′orientation; c) a first promoter adjacent to the 5′ end of the acceptorsequence-specific recombinase target site; and d) an acceptor-partialselectable marker, wherein the acceptor-partial selectable marker iscapable of recombining with a donor-partial selectable marker from adonor construct (or first donor fragment, once the donor construct isresolved) so creating a recombinant-functional selectable marker in afinal desired recombination construct. As in the donor construct, theacceptor construct is not limited by the nature of the sequence-specificrecombinase target site employed, and in preferred embodiments thesequence-specific recombinase target site may be selected from the groupconsisting of loxP, loxP2, loxP511, loxP514, loxB, loxC2, loxL, loxR,loxΔ86, loxΔ117, loxP3, loxP23, att, dif, and frt. The acceptorsequence-specific recombinase target site from the acceptor constructdoes not have to be identical to those on the donor construct; however,the sequence-specific recombinase target sites on the acceptor and donorconstructs must be able to recombine with each other.

In a preferred embodiment, the acceptor-partial selectable marker is asecond promoter, wherein the second promoter is oriented such that its5′ to 3′ orientation is opposite that of the acceptor sequence-specificrecombinase target site and the first promoter, and wherein the 3′ endof the second promoter is adjacent to the 3′ end of the acceptorsequence-specific recombinase target site.

The acceptor construct is not limited by the nature of the origin ofreplication employed. A variety of origins of replication are known inthe art and may be employed on the acceptor nucleic acid constructs ofthe present invention. Those skilled in the art know that the choice oforigin depends on the application for which the acceptor construct isintended and/or the host strain in which the construct is to bepropagated. In the case of the acceptor construct, the origin ofreplication is chosen appropriately such that both the acceptorconstruct and the final desired recombination construct will be able toreplicate in the given host cell.

The acceptor construct also is not limited by the nature of thepromoters employed. Those skilled in the art know that the choice of thepromoter depends upon the type of host cell to be employed forexpressing a gene(s) under the transcriptional control of the chosenpromoter. A wide variety of promoters functional in viruses, prokaryoticcells and eukaryotic cells are known in the art and may be employed inthe acceptor nucleic acid constructs of the present invention. In apreferred embodiment of the invention, the donor construct contains agene or DNA sequences of interest and when the donor constructrecombines with the acceptor construct, the first promoter of theacceptor construct is positioned such that it will drive expression ofthe gene or DNA sequences of interest. Thus, a promoter capable ofdriving the gene or DNA sequences of interest should be chosen for thefirst promoter. Further, in a preferred embodiment of the presentinvention, the acceptor-partial selectable marker is a promoter capableof driving the expression of the donor-partial selectable marker ORFfrom the donor construct (e.g., the promoter for the ampicillin genefrom the plasmid pUC19) or a viral promoter including, but not limitedto, the T7, T3, and Sp6 promoters.

In yet another preferred embodiment of the acceptor construct, theacceptor construct additionally includes a DNA sequence encoding apeptide affinity domain or peptide tag sequence, wherein the affinitydomain or tag sequence is 3′ of the first promoter and 5′ of theacceptor sequence-specific recombinase target site, such that theexpression of the affinity domain or tag sequence is under control ofthe first promoter, and such that it is in the same translational frameas the acceptor sequence-specific recombinase target site. The presentinvention is not limited by the nature of the affinity domain or tagsequence employed; a variety of suitable affinity domains are known inthe art, including glutathione-S-transferase, the maltose bindingprotein, protein A, protein L, polyhistidine tracts, etc.; and tagsequences include, but are not limited to the c-Myc Tag, the HA Tag, theFLAG tag, Green Fluorescent Protein (GFP), etc.

In another preferred embodiment of the acceptor construct, the acceptorconstruct further includes an acceptor-functional selectable marker. Thepresent invention is not limited by the nature of theacceptor-functional selectable marker chosen and the selectable markergene may result in positive or negative selection. In a preferredembodiment, the acceptor-functional selectable marker gene is selectedfrom the group consisting of the chloramphenicol resistance gene, theampicillin resistance gene, the tetracycline resistance gene, thekanamycin resistance gene, the streptomycin resistance gene and the sacBgene.

In addition to one or more of the above described components, theacceptor vectors may include a number of additional components thatimpart specific function to the expression vectors that are producedfrom the acceptor vector according to the subject methods. Additionalelements that may be present on the subject acceptor vectors include,but are not limited to: (a) elements requisite for generating vectorssuitable for use in yeast two hybrid expression assays, e.g., a GAL4activation domain coding sequence, a GAL4 DNA-binding domain codingsequence, (as found in pLP-GADT7 and pLP-GBKT7 shown in FIGS. 3A & 3B);(b) elements necessary for study of the localization of a protein in acell, e.g., tagging elements such as fluorescent protein codingsequences, such as the GFP coding sequences (as found in pLP-EGFP-C1,pLP-ECFP-C1 and pLP-EYFP-C1 shown in FIGS. 3C to 3E); (c) elementsnecessary for constitutive, bicistronic expression in mammalian cells,e.g., IRES sites, in combination with selectable markers, e.g.antibiotic resistance, fluorescent protein, etc. (as found inpLP-IRESneo and pLP-IRES2-EGFP shown in FIGS. 3F to 3G); (d) elementsnecessary for inducible expression of the gene of interest on anexpression vector, e.g. inducible promoters such as the tet-responsivepromoter, etc. (as illustrated by pLP-TRE2, pLP-ProTet and pLP-RevTRE,shown in FIGS. 3H, 3I and 3K); (e) elements that provide for retroviralexpression vectors, e.g., as found in pLP-LNCX and pLP-RevTre shown inFIGS. 31 and 3J; and the like.

Also provided is an alternative acceptor construct embodiment that canbe used with the alternative donor vector described above. In thisembodiment, the alternative acceptor construct includes: a) an origin ofreplication; b) a first sequence-specific recombinase target site and asecond sequence-specific recombinase target site each having a 5′ and a3′ orientation, wherein said first and second sequence-specificrecombinase target sites have the same 5′ to 3′ orientation and wheresaid first and second sequence-specific recombinase target sites canrecombine with each other and with the sequence-specific recombinasetarget site of said alternative donor construct; c) a first promoterelement having the same 5′ to 3′ orientation as the sequence-specificrecombinase target sites and wherein said first promoter element ispositioned at the 5′ end of said second sequence-specific recombinationtarget site; and d) an acceptor partial selectable marker elementwherein said acceptor partial selectable marker element is capable ofrecombining with said donor partial selectable marker element from saidalternative donor construct to create a functional selectable markerelement in the final recombination construct. In a preferred embodimentof this alternative embodiment, said acceptor partial selectable markerelement is a second promoter having a 5′ and 3′ end, wherein said secondpromoter is oriented such that its 5′ to 3′ orientation is opposite tothat of said acceptor sequence-specific recombination sites and saidfirst promoter element, and wherein the 3′ end of said second promoteris adjacent to the 3′ end of the first sequence-specific recombinationsite. Also in a preferred embodiment of the alternative embodiment ofthe acceptor construct, the acceptor construct additionally comprises aDNA sequence encoding a peptide affinity domain or peptide tag sequence,wherein said affinity domain is under control of the said first promoterelement and is in the same translational frame as the secondsequence-specific recombinase site. Also, a preferred embodiment of thealternative embodiment of the acceptor construct further comprises afunctional selectable marker gene.

Expression Vector Generation with a Recombinase

As mentioned above, in the subject methods the donor and acceptorvectors are contacted with a recombinase under conditions sufficient forsite specific recombination to occur, specifically under conditionssufficient for a recombinase mediated recombination event to occur thatproduces the desired expression vector, where expression vectorproduction is accomplished without cutting or ligation of the donor andacceptor vectors with restriction endonucleases and nucleic acidligases. The contact may occur under in vitro or in vivo conditions, asis desired and/or convenient.

In many embodiments, an aqueous reaction mixture is produced bycombining the donor and acceptor vectors and the recombinase with waterand other requisite and/or desired components to produce a reactionmixture that, under appropriate conditions, results in production of thedesired expression vector. The various components may be combinedseparately or simultaneously, depending on the nature of the particularcomponent and how the components are combined. Conveniently, thecomponents of the reaction mixture are combined in a suitable container.The amount of donor and acceptor vectors that are present in thereaction mixture are sufficient to provide for the desired production ofthe expression vector product, where the amounts of donor and acceptorvector may be the same or different, but are in many embodimentssubstantially the same if not the same. In many embodiments, the amountof donor and acceptor vector that is present in the reaction mixtureranges from about 50 ng to 2 ug, usually from about 100 ng to 500 ng andmore usually from about 150 ng to 300 ng, for a reaction volume rangingfrom about 5 μl to 1000 μl, usually from about 10 μl to 50 μl.

The recombinase that is present in the reaction mixture is one thatprovides for recombination of the donor and acceptor vectors, i.e. onethat recognizes the recombinase recognition sites on the donor andacceptor vectors. As such, the recombinase employed will vary, whererepresentative recombinases include, but are not limited to:recombinases, transposes and integrases, where specific recombinases ofinterest include, but are not limited to: Cre recombinase (the cre genehas been cloned and expressed in a variety of hosts, and the enzyme canbe purified to homogeneity using standard techniques known in theart—purified Cre protein is available commercially from Novagen); FLPrecombinase of S. cerevisiae that recognizes the frt site; Intrecombinase of bacteriophage Lambda that recognizes the att site; xerCand xerD recombinases of E. coli, which together form a recombinase thatrecognizes the dif site. the Int protein from the Tn916 transposon; theTn3 resolvase, the Hin recombinase; the Cin recombinase; theimmunoglobulin recombinases; and the like. While the amount ofrecombinase present in the reaction mixture may vary depending on theparticular recombinase employed, in many embodiments the amount rangesfrom about 0.1 units to 1250 units, usually from about 1 unit to 10units and more usually from about 1 unit to 2 units, for the abovedescribed reaction volumes. The aqueous reaction mixture may includeadditional components, e.g., a reaction buffer or components thereof,e.g., buffering compounds, such as Tris-HCl; MES; sodium phosphatebuffer, sodium acetate buffer; and the like, which are often present inamounts ranging from about 10_mM to 100 mM, usually from about 20 mM to_(—)50 mM; monovalent ions, e.g., sodium, chloride, and the like, whichare typically present in amounts ranging from about 10 mM to 500 mM,usually from about 30 mM to 150 mM; divalent cations, e.g., magnesium,calcium and the like, which are often present in amounts ranging fromabout 1 mM to 20 mM, usually from about 5 mM to 10 mM; and othercomponents, e.g., BSA, EDTA, spermidine and the like; etc (where theabove amount ranges are provided for the representative reaction volumesdescribed above). As the reaction mixtures are aqueous reactionmixtures, they also include water.

The subject reaction mixtures are typically prepared at temperaturesranging from about 0-4° C., e.g., on ice, to minimize enzyme activity.Following reaction mixture preparation, the temperature of the reactionmixture is typically raised to a temperature that provides for optimumor maximal recombinase activity, and concomitantly expression vectorproduction. Often, in this portion of the method the temperature will beraised to a temperature ranging from about 4° C. to 37° C., usually fromabout 10° C. to 25° C., where the mixture will be maintained at thistemperature for a period of time sufficient for the desired amount ofexpression vector production to occur, e.g., for a period of timeranging from about 5 mins to 60 mins, usually from about 10 mins to 15mins. Following the incubation period, the reaction mixture is subjectedto conditions sufficient to inactivate the recombinase, e.g., thetemperature of the reaction mixture may be raised to a value rangingfrom about 65° C. to 70° C. for a period of time ranging from about 5mins to 10 mins.

Alternatively, contact of the donor and acceptor vectors with therecombinase may occur in vivo, where the donor and acceptor vectors areintroduced in a suitable host cell that expresses a recombinase. In thisembodiment, the recombination between the donor and acceptor vectors maybe accomplished in vivo using a host cell that transiently orconstitutively expresses the appropriate site-specific recombinase(e.g., Cre recombinase expressed in the bacterial strain BNN132,available from CLONTECH). pDonor and pAcceptor, i.e., the donor andacceptor vectors respectively, are co-transformed into the host cellusing a variety of methods known in the art (e.g., transformation ofcells made competent by treatment with CaCl₂, electroporation, etc.).The co-transformed host cells are grown under conditions which selectfor the presence of the recombinant-functional selectable marker createdby recombination of pDonor with the pAcceptor (e.g., growth in thepresence of chloramphenicol when the pDonor vector contains all or partof the chloramphenicol resistance gene open reading frame and pAcceptormay also contain a promoter necessary for expression of thechloramphenicol open frame). Plasmid DNA is isolated from host cellswhich grow in the presence of the selective pressure and is subjected torestriction enzyme digestion to confirm that the desired recombinationevent has occurred.

The present invention also provides a method for the in vitrorecombination of nucleic acid constructs, comprising the steps of: a)providing i) a donor nucleic acid construct comprising a donor-partialselectable marker, two donor sequence-specific recombinase target siteseach having a defined 5′ to 3′ orientation and wherein the donorsequence-specific recombinase target sites are placed in the donorconstruct such that they have the same 5′ to 3′ orientation, and aunique restriction enzyme site or polylinker, the restriction enzymesite or polylinker being located 3′ of the first-donor sequence-specificrecombinase target site and 5′ of the second-donor sequence-specificrecombinase target site; (ii) an acceptor nucleic acid constructcomprising an origin of replication, an acceptor sequence-specificrecombinase target site having a defined 5′ to 3′ orientation, a firstpromoter adjacent to the 5′ end of the acceptor sequence-specificrecombinase target site, and an acceptor-partial selectable marker,wherein the acceptor-partial selectable marker is capable of recombiningwith the donor-partial selectable marker from the donor construct tocreate a recombinant-functional selectable marker in a final desiredrecombination construct; b) contacting the donor and acceptor constructsin vitro with a site-specific recombinase under conditions such that thedesired donor fragment recombines with the acceptor construct to form afinal desired recombination construct.

The present invention further provides a method for the recombination ofnucleic acid constructs in a host, comprising the steps of: a) providingi) a donor nucleic acid construct comprising a donor-partial selectablemarker, two donor sequence-specific recombinase target sites each havinga defined 5′ to 3′ orientation and wherein the donor sequence-specificrecombinase target sites are placed in the donor construct such thatthey have the same 5′ to 3′ orientation, and a unique restriction enzymesite or polylinker, the restriction enzyme site or polylinker located 3′of the first-donor sequence-specific recombinase target site and 5′ ofthe second-donor sequence-specific recombinase target site; (ii) anacceptor nucleic acid construct comprising an origin of replication, anacceptor sequence-specific recombinase target site having a defined 5′to 3′ orientation, a first promoter adjacent to the 5′ end of theacceptor sequence-specific recombinase target site, and anacceptor-partial selectable marker, wherein the acceptor-partialselectable marker is capable of recombining with the donor-partialselectable marker from the donor to create a recombinant-functionalselectable marker in a final desired recombination construct; and iii) ahost cell expressing a site-specific recombinase; b) introducing thedonor and acceptor constructs into the host cell under conditions suchthat the desired donor fragment recombines with the acceptor constructto form the final desired recombination construct which is capable ofimparting the ability to the host cell to grow in selective growthmedium.

The above methods of producing expression vectors can be employed torapidly produce a plurality of different expression vectors that aredistinct from each other but carry the same coding sequence of interestfrom a single, original type of donor vector. In other words, thesubject methods can be used to rapidly clone a nucleic acid of interestfrom an initial vector into a plurality of expression vectors. Byplurality is meant at least 2, usually at least 5, and more usually atleast 10, where the number may be as high as 20, 96 or more. The methodscan be performed by one person in a period of time that is a fraction ofwhat it would take by that person of skill in the art to produce thesame number and variety of expression vectors using traditional cuttingand ligation protocols, where the increase in efficiency obtained by thesubject methods is at least about 6 fold, usually at least about 15 foldand more usually at least about 30 fold.

The Resultant Expression Vector

The above steps result in the production of an expression vector fromdonor and acceptor vectors, and more specifically from a portion of oneof these vectors and the entirety of the other of these vectors, e.g.,from a portion of the donor vector and the entirety of the acceptorvector, where by portion is meant the part of the donor vector that lies3′ of the first donor sequence-specific recombinase site and 5′ of thesecond donor sequence-specific recombinase site. The size of theexpression vector may vary, depending on the nature of the vector. Wherethe vector is a plasmid, the size of the expression vector may rangefrom about 3 kb to 20 kb, usually from about 4 kb to 8 kb.

The resultant expression vector is characterized in that it includes tworecombinase recognition sites, i.e., a first and second recombinaserecognition site, oriented in the same direction. The distance betweenthe first and second recombinase sites, specifically the distancebetween the 3′ end of the first recombinase site and the 5′ end of thesecond recombinase site, ranges in many embodiments from about 100 bp to100 kb, usually from about 500 bp to 20 kb, depending on whether thecoding sequence of a protein of interest or just a restrictionsite/multiple cloning site, is present between the first and secondrecombinase recognition sites. The portion of the vector that lies inthis inter recombinase region, i.e. 3′ of the first recombinase site and5′ of the second recombinase site, typically makes up from about 2% to85%, usually from about 20% to 60% of the entire expression vector.

In many embodiments, the expression vector is further characterized inthat 5′ of the first recombinase site is a first promoter, 3′ of thefirst recombinase site is at least one restriction site; and the secondrecombinase site located inside a functional selectable marker, i.e., itis flanked by disparate portions or sub-parts of a selectable markerexpression module or cassette (e.g., a promoter and a coding sequence),where the second recombinase site is present between the two sub-partsof the selectable marker in a manner such that the selectable marker isfunctional, i.e., the coding sequence of the selectable marker isexpressed. In other words the expression vector includes a selectablemarker expression cassette or module made up of a promoter and codingsequence that flank the second recombinase site. In many embodiments,the second recombinase site is flanked by a promoter on its 3′ end and acoding sequence of the selectable marker on its 5′ end. In thisembodiment, the first and second promoters, located 5′ of the firstrecombinase site and 3′ of the second recombinase site, respectively,are oriented in opposite directions.

The expression vector is further characterized by having at least onerestriction site, and generally a multiple cloning site, located betweenthe first and second recombinase sites. In many embodiments, locatedbetween the first and second recombinase sites, and flanked by tworestriction sites, which may or may not be the same, is a nucleic acidof interest, i.e., gene of interest, that includes a coding sequence fora protein of interest whose expression from the expression vector isdesired. In these embodiments, the first promoter 5′ of the firstrecombinase site and the coding sequence for the protein of interest arearranged on either side of the first recombinase site such that theyform an expression module or cassette that expresses the encodedprotein, i.e., the coding sequence and first promoter flank the firstrecombinase site in manner such that they are operably linked.

In addition to the above features, the expression vector furtherincludes at least one origin of replication that provides forreplication in the host or hosts into which it is placed or transformedduring use. Origins of replication of interest include, but are notlimited to, those described above in connection with the donor andacceptor vectors.

In a specific embodiment, the expression vector or final construct ischaracterized as follows—this final desired recombination constructcomprises, in operable 5′ to 3′ order: a) a first promoter; b) afirst-recombinant sequence-specific recombinase target site, wherein the5′ end of the first-recombinant sequence-specific recombinase targetsite is derived from the 5′ end of the acceptor sequence-specificrecombinase target site from the acceptor and the 3′ end of thefirst-recombinant sequence-specific recombinase target site is derivedfrom the 3′ end of the first-donor sequence-specific recombinase targetsite of the donor construct; c) a unique restriction enzyme site orpolylinker; d) the donor-partial selectable marker; e) asecond-recombinant sequence-specific recombinase target site locatedwithin the recombinant-functional selectable marker gene and adjacent tothe donor-partial selectable marker and the acceptor-partial selectablemarker, wherein the 5′ end of the second-recombinant sequence-specificrecombinase target site is derived from the 5′ end of the second-donorsequence-specific recombinase target site from the donor construct andthe 3′ end of the second-recombinant sequence-specific recombinasetarget site is derived from the 3′ end of the acceptor sequence-specificrecombinase target site of the acceptor construct; f) theacceptor-partial selectable marker, wherein the acceptor-partialselectable marker adjoins the donor-partial selectable marker to producea newly-created recombinant-functional selectable marker; and, g) anorigin of replication.

In a preferred embodiment, the final desired recombination productcontains a gene or DNA sequence of interest inserted into the uniquerestriction enzyme site or polylinker such that the gene or DNA sequenceof interest is under the control of the first promoter. In such anembodiment, the gene or DNA sequence of interest is joined to the 3′ endof the first-recombinant sequence-specific recombinase target site suchthat a functional transcriptional unit is formed so that the gene or DNAsequence of interest is expressed as a protein driven by the firstpromoter of the acceptor construct. hi a more preferred embodiment, thegene of interest is joined to the 3′ end of the first-recombinantsequence-specific recombinase target site such that a functionaltranslational reading frame is created wherein the gene or DNA sequenceof interest is expressed as a fusion protein with an affinity domain ortag sequence derived from the acceptor plasmid and under the expressioncontrol of the first promoter of the acceptor construct.

In another preferred embodiment, the final desired recombinationconstruct further comprises an acceptor-functional selectable markergene derived from the acceptor construct. If an acceptor-functionalselectable marker gene is present in addition to the newly-createdrecombinant-functional selectable marker, the acceptor-functionalselectable marker is a different selectable marker from thenewly-created recombinant-functional selectable marker. The presentinvention should not be limited by the nature of the selectable markergenes chosen; the marker genes may result in positive or negativeselection and may be chosen from the group including, but not limitedto, the chloramphenicol resistance gene, the ampicillin resistance gene,the tetracycline resistance gene, the kanamycin resistance gene, thestreptomycin resistance gene, the strA gene and the sacB gene.

Utility

The subject methods find use in a variety of different applications,where such applications are generally those protocols and methods inwhich the transfer of a nucleic acid of interest from one vector toanother, e.g., the cloning of a nucleic acid from an initial vector intoa final vector, is desired. As such, the subject methods areparticularly suited for use in cloning nucleic acids of interest,including whole libraries, from an initial vector into an expressionvector, where the expression vector may be functionalized to express thepolypeptide or protein encoded by the nucleic acid of interest locatedon it in a variety of different desired environments and/or underdesired conditions, e.g., in a cell of interest, in response to aparticular stimulus, tagged by a detectable marker, etc.

As such, the expression vectors produced by the subject methods find usein a variety of different applications, including the study ofpolypeptide and protein function and behavior, i.e., in thecharacterization of a polypeptide or protein, either known or unknown;and the like. In the broadest sense, the subject methods findapplication in any method where traditional digestion and ligationprotocols are employed to transfer or clone a nucleic acid from onevector to another, e.g., cloning digestion and ligation protocols, wherethe expression vectors produced by the subject methods find use inresearch applications, as well as other applications, e.g., proteinproduction applications, therapeutic applications, and the like.

Systems

Also provided are systems for use in practicing the subject methods. Thesubject systems at least include a donor vector and an acceptor vectoras described above. In addition, the subject systems may include arecombinase which recognizes the recombinase sites present on the donorand acceptor vectors. The systems may also include, where desired, ahost cell, e.g., in in vivo methods of expression vector production, asdescribed above. Other components of the subject systems include, butare not limited to: reaction buffer, controls, etc.

Libraries

Also provided are nucleic acid libraries cloned into donor and/oracceptor vectors of the subject invention. These nucleic acid librariesare made up of a plurality of individual donor/acceptor vectors whereeach distinct constituent member of the library has a different nucleicacid portion or component, e.g., genomic fragment, cDNA, of an originalwhole nucleic acid library, i.e., fragmented genome, cDNA collectiongenerated from the total or partial mRNA of an mRNA sample, etc. Inother words, the libraries of the subject invention are nucleic acidlibraries cloned into donor or acceptor vectors according to the subjectinvention, where the nucleic acid libraries include, but are not limitedto, genomic libraries, cDNA libraries, etc. Specific donor/acceptorlibraries of interest include, but are not limited to: Human Brain PolyA+RNA; Human Heart Poly A+RNA; Human Kidney Poly A+RNA; Human Liver PolyA+RNA; Human Lung Poly A+RNA; Human Pancreas Poly A+RNA; Human PlacentaPoly A+RNA; Human Skeletal Muscle Poly A+RNA; Human Testis Poly A+RNA;Human Prostate Poly A+RNA and the like. With donor libraries accordingto the subject invention, the subject methods permit the rapid exchangeof either individual clones of interest, groups of clones or potentiallyan entire cDNA library to a variety of expression vectors. The cDNAlibrary is generated using a pDonor construct as the cloning vector (apDonor library, e.g., pDNR-Lib as shown in FIG. 2D). The entire librarymay then be transferred (using either an in vitro or an in vivorecombination reaction) into any expression vector modified to containan acceptor sequence-specific recombinase target site (e.g., a lox site)(i.e., an acceptor construct). This solves an existing problem in theart, in that there is no way, using existing vector systems, to exchangethe inserts in a library made in one expression vector en masse (i.e.,as an entire library) to a different expression vector.

Kits

Also provided are kits for use in practicing the subject methods. Thesubject kits at least include at least one donor vector and arecombinase that recognizes the recombinase sites of the donor vector.The subject kits may further include other components that find use inthe subject methods, e.g., acceptor vectors; reaction buffers, positivecontrols, negative controls, etc.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Construction of a pDonor Construct

This example describes a donor construct, the pD3 vector, whichcontained two loxP sites, a polylinker, a chloramphenicol resistancegene (Cm^(R)) open reading frame lacking a promoter, a standard originof replication (derived from pUC19) and an ampicillin resistance gene(Amp^(R)) with its associated promoter. If a gene of interest iscontained within pD3, any number of plasmid expression constructscontaining this gene of interest can be constructed rapidly (e.g.,within a single day). The expression constructs (the acceptor constructor the pAcceptor used in this example was pCMVmycloxP (described below))contained a sequence-specific recombinase target site, a promotercapable of expressing a gene of interest, an antibiotic resistance geneother than chloramphenicol (e.g., ampicillin), and a promoter positionedsuch that upon recombination of the pAcceptor with pD3, the promoterdrove expression of the Cm^(R) open reading frame from pD3.

Using a site-specific recombinase, Cre, a fragment of the initial donorconstruct encoding the gene of interest and the Cm^(R) open readingframe recombined into the pAcceptor construct at its loxP site,resulting in the production of a vector in which the fragment of pD3having the Cm^(R) open reading frame was placed under the control of thesecond promoter on pCMVmycloxP. The recombination of pD3 and pCMVmycloxPto form the final desired recombinant construct was selected for by theability of cells transformed with the constructs to grow in the presenceof chloramphenicol.

The plasmid backbone used to generate pD3 was the pUC19 plasmid. Thus,the origin of replication and the second selectable marker gene of pD3were the pUC origin of replication and the pUC Ampicillin resistancegene, respectively. This base vector further was derived to generate pD3as follows:

1. pUC19 was digested with AatII and SapI to remove the regioncontaining the LacZ gene and polylinker (nucleotides 2617-2686; 1-690).Into the remaining fragment were cloned two double-strandedoligonucleotides made by annealing the following two pairs of singlestranded oligonucleotides:

LoxP1-up: 5′-CGCGGCCGCATAACTTCGTATAGCATACATTATACGAAGTTATCAGTCGACG-3′(SEQ ID No. 1);

LoxP1-down:5′-AATTCGTCGACTGATAACTTCGTATAATGTATGCTATACGAAGTTATGCGGCCGCGACGT-3′ (SEQID No. 2);

LoxP2-up: 5′-AATTCGGATCCATAACTTCGTATAGCATACATTATACGAAGTTATGCGGCC-3′ (SEQID No. 3);

LoxP2-down: 5′-AGCGGCCGCATAACTTCGTATAATGTATGCTATACGAAGTTATGGATCCG-3′(SEQ ID No. 4).

The first pair of oligonucleotides encoded overhangs for AatII and EcoRIand the second pair encoded overhangs for EcoRI and SapI. These twopairs of oligos were thus ligated at their common EcoRI overhang andwere subsequently able to be cloned into the AatII and SapI-digestedpUC19 DNA. In the process, the SapI site was lost. In addition to therestriction sites mentioned, the LoxP1-up/down oligonucleotide pair alsoencoded a NotI site (GCGGCCGC) (SEQ ID No. 5). Similarly, theLoxP2-up/down pair also encoded a NotI site and a BamHI site (GGATCC)(SEQ ID No. 6). This first construct is called pD1.

2. pD1 was digested with BamHI and EcoRI, and a PCR fragment encodingthe chloramphenicol resistance gene open reading frame (Cm^(R)) andtermination sequence (nucleotides: 1932-1115, complement of the vectorpProTet.E121, available from CLONTECH) was inserted using an EcoRI siteand a BglII site engineered into the following reverse and forward PCRprimers, respectively:

Cm^(R)-fwd: 5′-ATGCTTGATACTAGATCTTTCAGGAGCTAAGGAAGCTA-3′ (SEQ ID No. 7);

Cm^(R)-rev: 5′-ATGCTGAATTCTGGATCCTGGTCATGACTAGTGCTT GG-3′ (SEQ ID No.8).

This resulted in the placement of the Cm^(R) open reading frame adjacentto the 5′ end of the second loxP site but in the reverse 5′ to 3′orientation. In addition, the original BamHI site in pD1 was destroyedand a new BamHI site was created adjacent to the EcoRI site. This vectoris called pD2.

3. pD2 was cut with NotI and religated, so as to invert the orientationof the cassette encoding the LoxP sites and the Cm^(R) open readingframe with respect to the ampicillin resistance selectable marker in thepUC19 backbone. This construct was called pD3.

4. pD3 is digested with EcoRI and BamHI and a PCR fragment encoding theT1 termination sequence (nucleotides 232-343 of pPROTet.E121) isinserted by standard methods. The resultant plasmid is pD4.

5. pD4 is restricted with EcoRI and BamHI and a PCR fragment encodingthe SV40 polyadenylation sequence is cloned into the vector. Theresultant vector is pD5.

6. PD5 is digested with BamHI and SalI and an oligo encoding a multiplecloning site is cloned into the BamHI and SalI sites to generate thefinal basic donor construct.

Example 2 Construction of pCMV-myc-LoxP

Acceptor constructs for the donor recombination system are generallyexpression vectors which have been modified by the insertion of a loxPor other sequence-specific recombinase target site and a prokaryoticpromoter in a position 3′ of the sequence-specific recombinase targetsite and oriented such as to direct transcription through thesequence-specific recombinase target site. It is also possible toutilize readthrough transcription from other promoters in the expressionvector, provided that their orientation and distance from the loxP siteis such that they can drive expression of the donor partial-selectablemarker gene upon recombination of the acceptor vector with the desiredfragment of the donor vector. The presence of a loxP site on theacceptor construct permitted the rapid subcloning or insertion of thegene interest contained within the pDonor vector to generate a finalrecombination construct capable of expressing the gene of interest. Theacceptor construct may encode a protein domain such as an affinitydomain or sequence tag including, but not limited to,glutathione-S-transferase (GST), maltose binding protein (MBP), proteinA, protein L, a polyhistidine tract, the c-Myc Tag, the HA tag, the FlagTag, Green Flourescence protein, etc. A variety ofcommercially-available expression vectors encoding such affinity domainsand tag sequences are known in the art. When the acceptor constructencodes an affinity domain, a fusion protein comprising the affinitydomain and the protein of interest is generated when the proper pDonorfragment and the acceptor constructs are recombined.

To generate final recombination constructs having the appropriatetranscriptional fusions, a sequence-specific recombinase target site wasplaced after (i.e., downstream of) the start of transcription in theacceptor construct. In designing the oligonucleotide comprising thesequence-specific recombinase target site, care was taken to avoidintroducing a start codon (the sequence “ATG”) which mightinappropriately initiate translation. Also, when generating a finalrecombination construct product having an appropriate translationalfusion between the acceptor-encoded protein domain and the donor-encodedgene of interest, care was taken to place the loxP site in the correctreading frame such that an open reading frame was maintained through thesequence-specific recombinase target site on pAcceptor, and the readingframe in the sequence-specific recombinase site on pAcceptor wasin-frame with the reading frame found in the first sequence-specificrecombinase target site contained within the pDonor construct. Inaddition, the oligonucleotide comprising the sequence-specificrecombinase target site on pAcceptor and the first sequence-specificrecombinase target site contained within the donor were designed toavoid the introduction of in-frame stop codons. The gene of interestcontained within the pDonor construct was cloned in a particular readingframe so as to facilitate the creation of the desired fusion protein.

Methods for modification of one expression vector are provided below toillustrate the creation of suitable pAcceptor constructs. The generalstrategy involves the generation of a linker containing asequence-specific recombinase target site by annealing two complementaryoligonucleotides. The annealed oligonucleotides form a linker havingsticky ends which were compatible with ends generated by restrictionenzymes whose sites are conveniently located in the parental expressionvector (e.g., within the polylinker of the parental expression vector).In addition, but not necessarily, a prokaryotic promoter was cloneddownstream of the sequence-specific recombinase target site with it's 5′to 3′ orientation such that it directed expression through thesequence-specific recombinase target site. pCMV-myc-LoxP is an examplepAcceptor construct. It was generated from pCMV-Myc (available fromCLONTECH) in the following way:

1. pCMV-Myc was digested with SfiI and BglII.

2. A double-stranded oligonucleotide encoding an overhang at its 5′ endcompatible with SfiI; a LoxP site; and an overhang at its 3′ endcompatible with BglII was generated by annealling the followingoligonucleotides together:

LoxPMyc-up: 5′-AGATAACTTCGTATAGCATACATTATACGAAGTTATA-3′ (SEQ ID No. 09);

LoxPMyc-down: 5′-GATCTATAACTTCGTATAATGTATGCTATACGAAGTTATCTCCA-3′ (SEQ IDNo.10).

This oligonucleotide was then cloned into the digested pCMVMyc vector togenerate pAccl.

3. The plasmid pAccl was then digested with BglII and NheI into which aPCR fragment encoding the ampicillin promoter from pUC19 (nucleotides:2620-2500, complement) was cloned. This fragment was generated usingappropriate primers encoding BamHI and NheI restriction sites asfollows:

AmpProFwd: 5′-ATGCTGGATCCAATATTATTGAAGCATTTATCA GG-3′ (SEQ ID No. 11);

AmpProRev: 5′-TCCATGCTGCTAGCACGTCAGGTGGCACTTTTCG-3′ (SEQ ID No. 12).

The resultant plasmid is pCMVMycLoxP, which is a basic Acceptor plasmidhaving a LoxP site and adjacent promoter to drive expression of the geneof interest in the same 5′ to 3′ orientation as the LoxP site and asecond promoter (acceptor partial selectable marker gene), oriented inthe reverse 5′ to 3′ direction as the LoxP site and placed adjacent tothe 3′ end of said LoxP site.

A similar strategy to generate other types of acceptor vectors will bereadily apparent to those skilled in the art. This strategy can beemployed to generate any number of pAcceptor constructs. It is onlynecessary to design the oligos and PCR primers with appropriaterestriction sites to match those in the polylinker of the construct tobe adapted.

Example 3 Generation of 10 Additional Acceptor Vectors

10 additional acceptor vectors, as described in FIGS. 3A-3J have beenmade as follows. The construction of these vectors was as follows:

Each parental vector used to generate the various acceptors was cut withtwo restriction enzymes that cut within the MCS of the vector, asdetailed below. Into these was inserted a PCR fragment of approx. 170 bpgenerated using various primers (described below) and pCMVMycLoxP, theacceptor molecule described above (see example 2 above) as a template.The primers are named either LoxP or AmpPro (to designate to which partof the template they are complementary) plus the name of the restrictionenzyme present in the 5′ end of the primer. These restriction sitesmatch the ones cut in the MCS of the vector to be modified. The fragmentgenerated in this PCR reaction encodes the LoxP site and the ampicillinpromoter from the pCMVMycLoxP acceptor.

List of the 10 vectors used and restriction sites and primers used inthe construction of each.

1. pGADT7: Cut with EcoRI and BamHI insert PCR fragment made withprimers LoxP-EcoRI and AmpPro (cut with enzymes EcoRI and BamHI)

2. pGBKT7: Cut with EcoRI and BamHI insert PCR fragment made withprimers LoxP-EcoRI and AmpPro (cut with enzymes EcoRI and BamHI)

3. pIRESneo: Cut with EcoRI and BamHI insert PCR fragment made withprimers LoxP-EcoRI and AmpPro (cut with enzymes EcoRI and BamHI)

4. pEGFP-C1: Cut with HindIII and BamHI insert PCR fragment made withprimers LoxP-HindIII and AmpPro-BamHI (cut with enzymes HindIII andBamHI)

5. pECFP-C1: Cut with HindIII and BamHI insert PCR fragment made withprimers LoxP-HindIII and AmpPro-BamHI (cut with enzymes HindIII andBamHI)

6. pEYFP-C1: Cut with HindIII and BamHI insert PCR fragment made withprimers LoxP-HindIII and AmpPro-BamHI (cut with enzymes HindIII andBamHI

7. pTRE2: Cut with SacII and BamHI insert PCR fragment made with primersLoxP-sacII and AmpPro-BamHI (cut with enzymes SacII and BamHI)

8. pRevTRE: Cut with HindIII and ClaI insert PCR fragment made withprimers LoxP-HindIII and AmpPro-ClaI (cut with enzymes HindIII and ClaI)

9. pLNCX: Cut with HindIII and ClaI insert PCR fragment made withprimers LoxP-HindIII and AmpPro-ClaI (cut with enzymes HindIII and ClaI

10. pIRES2-EGFP: Cut with EcoRI and BamHI insert PCR fragment made withprimers LoxP-EcoRI and AmpPro-BamHI (cut with enzymes EcoRI and BamHI)

Primers for amplification of insert, providing various restrictionenzyme ends (underlined) to enable cloning into above vectors.

1. LoxP-EcoRI

Sequence {5′-3′}: GATGCTGAATTCATAACTTCGTATAGCATACATTAT (SEQ ID NO:13) a36 mer

MW-O: 11025 MW-N: 11587 TM: 66.91666 Extinction Coef: 399 Mass(ug) perOD: 29.0401

2. AmpPro-BHI

Sequence {5′-3′}: AGTCTGGATCCACGTCAGGTGGCACTTTTCG (SEQ ID NO: 14) a 31mer

MW-O: 9512 MW-N: 9994 TM: 73.40323 Extinction Coef: 320 Mass(ug) per OD:31.23125

3. LoxP-HindIII

Sequence {5′-3′}: ATGCTAAGCTTCGATAACTTCGTATAGCATACATTAT (SEQ ID NO:15) a37 mer

MW-O: 11314 MW-N: 11892 TM: 67.85135 Extinction Coef: 406 Mass(ug) perOD: 29.29064

4. AmpPro-ClaI

Sequence {5′-3′}: AGTCTATCGATACGTCAGGTGGCACTTTTCG (SEQ ID NO:16) a 31mer

MW-O: 9511 MW-N: 9993 TM: 71.20968 Extinction Coef: 325 Mass(ug) per OD:30.74769

5. LoxP-NheI

Sequence {5′-3′}: TCCATGCTGCTAGCATAACTTCGTATAGCATACATTAT (SEQ ID NO:17)a 38 mer

MW-O: 11579 MW-N: 12173 TM: 69.63158 Extinction Coef: 405 Mass(ug) perOD: 30.05679

6. LoxP-SacII

Sequence {5′-3′}: TAGTACTCCGCGGATAACTTCGTATAGCATACATTAT (SEQ ID NO:18) a37 mer

MW-O: 11315 MW-N: 11893 TM: 69.68919 Extinction Coef: 401 Mass(ug) perOD: 29.65835

The primers were all made and PAGE purified by our regular supplier(Keystone labs) and were resuspended in water to a concentration of 100pmol/ul in water.

Example 4 In Vitro Recombination Using the pDonor Recombination System

The pDonor recombination system permits in vitro recombination of twoconstructs. FIG. 1 provides schematic showing the strategy employed forin vitro recombination. pDNR-1,2,3 represent typical pDonor constructswhich contains two loxP sites, a chloramphenicol resistance gene openreading frame which lacks a promoter, an origin of replication and anampicillin resistance marker. The desired Acceptor vector shown containsa loxP site, a prokaryotic promoter in opposite orientation to the loxPsite to drive the chloramphenicol open reading frame of pDNR-1,2,3, anampicillin resistance gene, a eukaryotic or prokaryotic promoter orfusion tag to permit expression of the gene of interest underappropriate conditions, and a pUC origin of replication.

To achieve generation of the expression vector from the donor andacceptor, the following were mixed together on ice in a standardeppendorf microcentrifuge tube: 0.5 μg pCMVmycloxP (representing theAcceptor vector); 0.5 μg pD3 (representing the Donor vector); 2 μl10×Cre reaction buffer (10×Cre reaction buffer contains: 500 mM Tris-HCl(pH 7.5) and 300 mM NaCl); 10 mM MgCl₂, 1 μl 20×BSA (20×BSA contains 2mg/ml BSA (NEB)); 25 Units Cre recombinase (Novagen); H₂O to 20 μltotal.

Once the reagents were mixed, the reaction was incubated for 15 mins at37° C. Following the reaction, the mixture was heated to 65° C. for 10mins to inactivate the Cre enzyme. Finally, an aliquot of the reactionmix was transformed to E. coli using standard methods (e.g.,electroporation), and the transformed bacteria selected on LB platescontaining 60 μg/ml Chloramphenicol.

Alternatively, the pDonor vector may be incubated with Cre alone underthe conditions described above; followed by purification of the fragmentbearing the gene of interest, e.g., by gel electrophoresis, andsubsequent recombination of the purified fragment into the pAcceptorvector, again according to the method above.

Example 5 The Use of Modified LoxP Sites to Increase Expression of theProtein of Interest

The pDonor and pAcceptor constructs employed in the pDonor recombinationsystem of the present invention are designed such that constructrecombination results in the introduction of a loxP site between thepromoter and the gene of interest. LoxP sites consist of two 13 bpinverted repeats separated by an 8 bp spacer region. Transcripts of thegene of interest produced from a pDonor-pAcceptor recombinationconstruct comprising a loxP site have two 13 nucleotide perfect invertedrepeats within the 5′ untranslated region (UTR) and have the potentialto form a stem-loop structure. In fact, this will occur in those caseswhere pAcceptor does not encode an affinity domain at the amino-terminusof the fusion protein. However, it is possible also to construct pDonorand pAcceptor constructs containing mutated loxP sequences. Mutated loxPsequences which comprise point mutations that create mismatches betweenthe two 13 bp inverted repeat sequences within the loxP sites and havemismatches at different positions in the inverted repeats located withina loxP site may be used. The suitability of any pair of mutated loxsites for use in the pDonor recombination system may be tested byreplacing the sequence-specific recombinase target sites in pDonor andpAcceptor with a site to be tested. The two modified vectors are thenrecombined in vitro as described in Example 3 and the recombinationreaction mixture is used to transform E. coli cells. The transformedcells are then plated on selective medium (e.g., Cm plates) in order todetermine the efficiency of recombination between the two mutated loxsites (Example 3). The efficiency of recombination between the twomutated lox sites is compared to the efficiency of recombination betweentwo wild-type loxP sites. It will be apparent to those skilled in theart that a similar strategy can be employed for the modification of frtsites when the FLP recombinase is employed for the recombination event,or other such recombinase sites as might be used. The frt site, like loxsites, contains two 13 bp inverted repeats separated by an 8 bp spacerregion.

Example 6 Alternative Conformations of pAcceptor and pDonor

The above-described constructs may be altered in the structuralorganization of their respective components, however, both constructsmust be altered such that following recombination, the donor-partial andacceptor-partial selectable markers comprise an intact,recombinant-functional selectable marker, and additionally, the firstpromoter is operably linked to the gene or DNA sequences of interest.For example, the invention could be done in a similar fashion asdescribed, except that the positions and orientations of thedonor-partial selectable marker on the Donor construct and theacceptor-partial selectable marker on the Acceptor construct areswitched. The final result of the recombination between the proper donorfragment (or first donor fragment) and the acceptor construct stillgenerates a recombinant-functional selection marker. Likewise, vectorssuch that the selection marker comprises two fragments and forms arecombinant-functional selectable marker in the final product by readingthrough the second sequence-specific recombinase target site are alsoincluded within this invention.

Example 7 Generation of Multiple Expression Constructs for Luciferase ina Single Day

Using the Donor recombination system, it is possible to transfer one ormany genes into multiple acceptor expression vectors at substantiallythe same time. To demonstrate this, the luciferase gene cloned into themultiple cloning site of pDNR-1, so generating pDNR-luc. The luciferasegene was then transferred from pDNR-luc into transferred to 10 differentacceptor vectors simultaneosly using the method described in EXAMPLE 4.To do this, each of ten individual acceptor vectors, as detailed inFIGS. 3A to 3J, was placed in an eppendorf tube in reaction buffer asdescribed in EXAMPLE 4. To each tube was then added 200 ng of pDNR-lucand 1 unit of cre recombinase. The reactions were incubated at 37° C.for 15 mins and then the Cre recombinase was inactivated by heating to65° C. for 10 mins. Each reaction was then transformed individually intoa separate aliquot of electro-competent DH5-alpha E. coli. These wereallowed to grow for 1 hour in the absence of selection and then wereplated out on selective agar plates containing 30 ug/ml chloramphenicoland 7% w/v sucrose. The following day, 3 colonies from eachtransformation were picked and grown-up for mini prep restriction digestanalysis to determine if the desired recombinant had been made. Of the30 clones analyzed in total (3 for each construct), 27 were correct,thus demonstrating that it is possible using the subject methods toreadily generate multiple expression constructs—in this example 10constructs—in a single day.

Example 8 Comparable Expression Levels for HEK 293 Cells TransfectedUsing Creator™ and Standard Vectors

To compare the expression level achievable with Creator vectors (i.e.,donor and acceptor vectors of the subject methods) to that generatedusing standard vectors, HEK 293 cells were transfected using the CalciumPhosphate method with either the pLP-EGFP-luc expression vectorgenerated as part of example 7 above, or the comparable vector madeusing traditional cloning methods—pEGFP-Luc (available from CLONTECH).24 hours after transfection, the level of fluorescence and the % ofcells transfected was determined by both fluorescence microscopy and byFACS analysis. The result showed that while there is some reduction inexpression associated with the Creator vectors, it is not a significanthindrance to adequate expression.

Example 9 Detection of Myc and Max Interaction by Yeast Two-hybridAnalysis

The interaction of myc and max proteins was tested by yeast two-hybridinteraction. To do this, relevant coding fragments of the human myc andmax genes were first cloned in to pDNR-I by standard restriction cloningmethods. These genes were then each transferred by use of the subjectmethods, as described in example 4 above, to both the pLP-GBKT7 (GAL4DNA binding domain—bait vector) and the pLP-GADT7 (GAL4 activationdomain—prey vector). AH109 yeast cells were then co-transformed witheither pLP-GADT7 and pLP-GBKT7 alone, or with the same two expressionvectors, but containing either myc or max. The yeast were then grown onselective medium lacking leucine and tryptophan in order to select forgrowth of yeast containing both constructs. The strength of theinteraction between the protein expressed in the bait and the preyconstructs was then determined using an alpha-galactosidase quantitativeassay and normalized for culture density, as described in the MATCHMAKERsystem III user manual (available from CLONTECH). In this way, it wasshown that myc and max interact well, but the homodimers do not.

Example 10 Inducible Expression of Luciferase in HeLa Cells UsingpLP-TRE-Luc

The expression construct pLP-TRE-Luc generated by recombination ofpLP-TRE and pDNR-Luc as described in Example 7 above, according to themethod in example 4 above, was transfected into HeLa Tet-Off cells(available from CLONTECH) using the geneporter lipofection kit(available from Gene Therapy Systems). The cells were then cultured for48 hrs in the presence of varying concentrations of doxycycline. Thecells were then harvested and assayed for luciferase activity. Theluciferase activity varied over several orders of magnitude from highlevel expression to background levels, dependent on the level ofDoxycycline present in the growth medium.

Example 11 High Level Luciferase Induction with pLP-TRE-luc andpLP-RevTre-Luc Compared to pTRE-luc

As described in example 8 above, the level of expression from Creatorvectors can be somewhat reduced when directly compared to comparableexpression vectors made using conventional cloning methods. It should benoted in this example that both basal and maximal levels of expressionare reduced. It is thought likely that this reduction is due toinhibition of RNA translation due to hairpins caused by the palindromiclox sites. As shown above, this reduction seems to have no significantlydetrimental effect on the functionality of any of the expression vectorstested. In this current example we further show that this reduction inexpression may actually be beneficial in the case of inducibleexpression. This is because the decrease in expression caused by the loxsites seems to more greatly affect low-level expression than it doesmaximal expression. For this reason, when fold induction of tetinducible vectors (either plasmid-based or retro viral) is comparedbetween standard vectors and creator vectors the fold induction seen ismuch greater in the case of the creator vectors. To demonstrate this,HeLa tet-off cells were transiently transfected with pTRE2-Luc orpLP-TRE2-luc, or stabily infected with pRevTRE-luc. Cells were thengrown for 48 hrs in the presence or absence of lug/ml doxycycline andthen assayed for luciferase activity. Both pLP-TRE2-luc andpLP-RevTRE-Luc were observed to show greater fold induction thanpTRE2-Luc.

Example 12 Construction of Other Acceptor Vectors

All of the following acceptor vectors are made simply by taking theparental vector and using PCR to insert a sequence encoding the loxPsite and the ampicillin promoter, into the MCS of the vector. Note thatthis sequence is present in all of the 10 acceptor vectors describedabove and can be obtained from them by PCR.

1: pLP-Shuttle, an acceptor vector for transferring genes of interestinto an adenoviral vector, is made by inserting the above sequence intothe NheI site and KpnI site of the pShuttle vector (available fromCLONTECH). This vector could itself be used, without a gene of interestto then transfer the loxP site and ampicillin promoter to adenoviralDNA, e.g., Adeno-X DNA (available from CLONTECH), so as to creator aadenovirus acceptor vector.

2: pLP-BacPAK9, an acceptor vector for transferring genes of interestinto an baculoviral vector, is made by inserting the above sequence intothe EcoRI site and BglII site of the pBacPAK9 vector (available fromCLONTECH). This vector could itself be used, without a gene of interestto then transfer the loxP site and ampicillin promoter to baculoviralDNA, e.g., Baculo Gold DNA (available from Pharmingen), so as to creatora baculovirus acceptor vector.

3: pLP-CMV-Myc, an acceptor vector providing constitutive mammalianexpression from the CMV promoter of myc epitope-tagged gene of interest,is made by inserting the above sequence into the SfiI site and BglIIsite of the pCMV-Myc vector (available from CLONTECH)

4: pLP-CMV-HA, an acceptor vector providing constitutive mammalianexpression from the CMV promoter of HA epitope-tagged gene of interest,is made by inserting the above sequence into the SfiI site and BglIIsite of the pCMV-HA vector (available from CLONTECH)

5: pLP-PROTet-6x(HN), an acceptor vector providing Tet-induciblebacterial expression of a 6x(HN)-tagged gene of interest, is made byinserting the above sequence into the HindIII and ClaI sites ofpPROTet.E133 (available from CLONTECH). Since this vector haschloramphenicol resistance, it should additionally be modified bychanging the chloramphenicol to ampicillin resistance.

It is evident from the above results and discussion that the subjectinvention provides an efficient method to transfer a nucleic acid from afirst vector to a second vector, where the subject methods do not employdigestion and ligation protocols. Advantages provided by the subjectinvention include: the ability to transfer or clone a nucleic acid ofinterest from a single donor into a variety of different expressionvectors at substantially the same time and in a known orientation andreading frame; the ability to readily identify successful clones; theability to transfer many different genes to one or more expressionvectors simultaneously; no longer needing to sequence the junctions ofthe transferred fragment and the expression vector or to resequence thegene transferred and the like. As such, the subject invention representsa significant contribution to the art.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

21 1 52 DNA Artificial Sequence synthetic oligonucleotide 1 cgcggccgcataacttcgta tagcatacat tatacgaagt tatcagtcga cg 52 2 60 DNA ArtificialSequence synthetic oligonucleotide 2 aattcgtcga ctgataactt cgtataatgtatgctatacg aagttatgcg gccgcgacgt 60 3 51 DNA Artificial Sequencesynthetic oligonucleotide 3 aattcggatc cataacttcg tatagcatac attatacgaagttatgcggc c 51 4 50 DNA Artificial Sequence synthetic oligonucleotide 4agcggccgca taacttcgta taatgtatgc tatacgaagt tatggatccg 50 5 8 DNAArtificial Sequence synthetic oligonucleotide 5 gcggccgc 8 6 6 DNAArtificial Sequence restriciton site 6 ggatcc 6 7 38 DNA ArtificialSequence pcr primer 7 atgcttgata ctagatcttt caggagctaa ggaagcta 38 8 38DNA Artificial Sequence pcr primer 8 atgctgaatt ctggatcctg gtcatgactagtgcttgg 38 9 37 DNA Artificial Sequence oligonucleotide 9 agataacttcgtatagcata cattatacga agttata 37 10 44 DNA Artificial Sequenceoligonucleotide 10 gatctataac ttcgtataat gtatgctata cgaagttatc tcca 4411 33 DNA Artificial Sequence primer 11 atgctggatc caatattatt gaagcatttatca 33 12 34 DNA Artificial Sequence primer 12 tccatgctgc tagcacgtcaggtggcactt ttcg 34 13 36 DNA Artificial Sequence primer 13 gatgctgaattcataacttc gtatagcata cattat 36 14 31 DNA Artificial Sequence primer 14agtctggatc cacgtcaggt ggcacttttc g 31 15 37 DNA Artificial Sequenceprimer 15 atgctaagct tcgataactt cgtatagcat acattat 37 16 31 DNAArtificial Sequence primer 16 agtctatcga tacgtcaggt ggcacttttc g 31 1738 DNA Artificial Sequence primer 17 tccatgctgc tagcataact tcgtatagcatacattat 38 18 37 DNA Artificial Sequence primer 18 tagtactccgcggataactt cgtatagcat acattat 37 19 105 DNA Artificial SequenceSynthetic Vector 19 ttatcagtcg acggtaccgg acatatgccc gggaattcctgcaggatccg ctcgagaagc 60 tttctagacc attcgtttgg cgcgcgggcc cagtaggtaagtgaa 105 20 105 DNA Artificial Sequence Synthetic Vector 20 ttatcagtcgactggtacca gacatatgcc cgggaattcc tgcaggatcc gctcgagaag 60 ctttctagaccattcgtttg gcgcgcgcat gcagtaggta agtga 105 21 101 DNA ArtificialSequence Synthetic Vector 21 ttatcagtcg actcggtacc gagcatatgc ccgggaattcctgcaggatc cgctcgaaag 60 cttatctaga cattcgtttg gcgcgcatgc atagtaggta a101

What is claimed is:
 1. A donor vector comprising: first and second recombinase recognition sites oriented in the same direction and flanking a partial selectable marker, wherein said first and second recombinase recognition sites are able to recombine with each other.
 2. The donor vector according to claim 1, wherein said partial selectable marker is a coding sequence.
 3. The donor vector according to claim 2, wherein said coding sequence is a coding sequence selected from the following group of genes: the chloramphenicol resistance gene, the ampicillin resistance gene, the tetracycline resistance gene, the kanamycin resistance gene, the streptomycin resistance gene and the SacB gene.
 4. The donor vector according to claim 1, wherein said recombinase recognition sites are selected from the group consisting of: lox sites, att sites, dif sites and frt sites.
 5. The donor vector according to claim 4, wherein said recombinase recognition sites are lox sites.
 6. The donor vector according to claim 1, wherein said donor vector further comprises a second functional selectable marker.
 7. The donor vector according to claim 6, wherein said second functional selectable marker is selected from the following group of genes: the chloramphenicol resistance gene, the ampicillin resistance gene, the tetracycline resistance gene, the kanamycin resistance gene, the streptomycin resistance gene and the SacB gene.
 8. The donor vector according to claim 1, wherein said donor vector further comprises a coding sequence for a protein of interest.
 9. The donor vector according to claim 1, wherein said donor vector is a plasmid, cosmid, bac, yac or virus.
 10. An acceptor vector comprising: a single recombinase recognition site located between a first promoter and a partial selectable marker, wherein said components are positioned such that, upon recombination of said acceptor vector with a donor vector that comprises a coding sequence for a protein of interest flanked by two recombinase recognition sites, an expression vector is produced that comprises an expression cassette made up of said first promoter and said coding sequence which flank a recombinase recognition site.
 11. The acceptor vector according to claim 10, wherein said partial selectable marker is a second promoter.
 12. The acceptor vector according to claim 11, wherein said second promoter is oriented in the opposite direction of said first promoter.
 13. The acceptor vector according to claim 10, wherein said partial selectable maker is a coding sequence for a selectable marker gene.
 14. The acceptor vector according to claim 10, wherein said recombinase recognition sites are selected from the group consisting of: lox sites, att sites, dif sites and frt sites.
 15. The acceptor vector according to claim 14, wherein said recombinase recognition site is a lox site.
 16. The acceptor vector according to claim 10, wherein said acceptor vector further comprises an origin of replication.
 17. The acceptor vector according to claim 10, wherein said acceptor vector is a plasmid, cosmid, bac, yac or virus.
 18. The acceptor vector according to claim 10, wherein said first promoter is operably linked to a tag encoding sequence.
 19. An expression vector comprising: (a) first and second recombinase recognition sites oriented in the same direction, wherein said first and second recombinase recognition sites recombine with each other; (b) an expression cassette for a protein of interest divided into two sub-parts that flank said first recombinase recognition site; and (c) a functional marker divided into two sub-parts that flank said second recombinase recognition site.
 20. The expression vector according to claim 19, wherein said recombinase recognition sites are selected from the group consisting of: lox sites, att sites, dif sites and frt sites.
 21. The expression vector according to claim 20, wherein said recombinase recognition sites are lox sites.
 22. The expression vector according to claim 19, wherein said two sub-parts of said expression cassette are a promoter and a coding sequence.
 23. The expression vector according to claim 22, wherein said coding sequence of said expression cassette is flanked by said first and second recombinase recognition sites.
 24. The expression vector according to claim 19, wherein said two sub-parts of said selectable marker are a promoter and a coding sequence of a selectable marker.
 25. The expression vector according to claim 24, wherein said selectable marker is selected from the following group of genes: the chloramphenicol resistance gene, the ampicillin resistance gene, the tetracycline resistance gene, the kanamycin resistance gene, the streptomycin resistance gene and the SacB gene.
 26. The expression vector according to claim 25, wherein said sub-parts of said expression cassette are a promoter and a coding sequence, and wherein said expression cassette promoter and said selectable marker promoter are oriented in opposite directions.
 27. The expression vector according to claim 19, wherein said vector is a plasmid, cosmid, bac, yac or virus.
 28. The expression vector according to claim 27, wherein said vector is a plasmid. 